The sterols of nine mature plant species in seven families ranging from the subphylum Lycopsida through the Filicopsida and the classes Gymnospermae and Angiospermae in the Pteropsida were structurally and stereochemically defined. Two plant categories were found. In the first, comprised by Dryopteris (Thelypteris) noveboracensis, Polystichum acrostichoides, Dennstaedtia punctilobula, Osmunda cinnamomea, Ginkgo biloba, Cucurbita pepo, and Kalmia latifolia, 24α-alkylsterols were dominant and were composed principally of 24α-ethylcholesterol (sitosterol) or (in Cucurbita pepo) 24α-ethyllathosterol and its trans-22-dehydro derivative (spinasterol). Depending on the species, small amounts of 24α-ethyl-trans-22-dehydrocholesterol (stigmasterol), 24α-methylcholesterol (campesterol), 24β-methylcholesterol (dihydrobrassicasterol, always less than campesterol), cholesterol, lathosterol, 24α-ethyllathosterol, 24ξ-methyllathosterol, trans-24-ethylidenelathosterol (Δ7-avenasterol), and (tentatively identified) 24-ethyl-24(25)-dehydrolathosterol were present. Spinacea oleracea was also confirmed as belonging to Category I, and, except as described in what follows, Category I represents all other configurationally investigated vascular plants. The second category of plants contained only 24β-ethylsterols. Only one species (Kalanchoe daigremontiana) belonging to the family Crassulaceae was found, but one other (the genus Clerodendrum in the family Verbenaceae) is already known. K. daigremontiana contained 25(27)-dehydroclionasterol (clerosterol) and 25(27)-dehydroporiferasterol. The primitive Lycopodium complanatum was intermediate between Categories I and II; the sterols with a 24-C2-group had only the 24α-configuration (sitosterol with some stigmasterol), but the principal sterols with a 24-C1-group (ergosterol and dihydrobrassicasterol) possessed the 24β-configuration. C. pepo seeds, which are already known to contain principally 24β-ethylsterols, contrast sharply with our finding that tissue (pericarp of the fruit) from the mature plant contains only 24α-ethylsterols. This apparent evolutionary recapitulation (Category II to I) during development coupled with statistical dominance of Category II plants among the algae and fungi and of Category I plants in the Tracheophytes, and the existence of an intermediate type in the species examined from the lower Tracheophyte (Lycopsida) lead logically to the conclusion that the 24α-alkyl structure, especially 24-α-ethyl-Δ5-sterols (sitosterol and the much rarer stigmasterol) constitutes the most highly evolved type of 24-alkylsterol. By inference from our knowledge of biosynthesis, corroborated by the spectrum of sterols found here, the pathway [through Δ24(25)-sterol] leading to 24α-alkylsterol appears to be higher than the pathway [through Δ25(27)-sterols] which leads to 24β-alkylsterols. The sterols of these and other plants were also found amenable to classification according to their nuclear unsaturation (Δ5, A; Δ7, B; and Δ5,7, C). Tracheophytes of Category A have been most frequently encountered, but C. pepo was shown to be of Category B throughout its ontogeny. While no Tracheophytes of pure Category C have been discovered, L. complanatum was shown to be of the mixed A-C-Type. Based on these facts and ideas, some previously suggested lines of botanical evolution are examined. The chemical data fail to verify a line from Magnoliales to the Cucurbitaceae, from Magnoliales through Theales to Ericales, nor from Ranales to Saxifragales. However, they are consonant with a relationship between Cucurbitaceae and Theales and between Rosales and Lamiales. Triterpenoids found in various of the families studied included cycloartenol and friedelin. The spectroscopic properties of the latter are described.