Heliothis zea (corn earworm), an insect that fails to synthesize sterols de novo, was reared on an artificial diet treated with 18 different sterol supplements. Larvae did not develop on a sterol-less medium. Δ5-Sterols with a hydrogen atom, a methylene group, an E- or Z-ethylidene group, or an α- or β-ethyl group (cholesterol, ostreasterol, isofucosterol, fucosterol, sitosterol, and clionasterol, respectively) at position C-24, and Δ5- sterols doubly substituted in the side chain at C-24 with an α-ethyl group and at C-22 with a double bond (stigmasterol) supported normal larval growth to late-sixth instar (prepupal: maturity). The major sterol isolated from each of these sterol treatments was cholesterol, suggesting that H. zea operates a typical 24-dealkylation pathway. The sterol requirement of H. zea could not be met satisfactorily by derivatives of 3β-cholestanol with a 913,19-cyclopropyl group, gem dimethyl group at C-4, a Δ5,7-bond or Δ8- bond, or by side-chain modified sterols that possessed a Δ(25(27))-24̄- ethYl group, Δ(23(24))-24-methyl group or 24-ethyl group, or Δ(24(25))-24- methyl or 24-ethyl group. The major sterol recovered from the larvae (albeit developmentally arrested larvae, treated with a nonutilizable sterol was the test compound. Sterol absorption was related to the degree of sterol utilization. The most effective sterols absorbed by the insect ranged from 27 to 66 μg per insect whereas the least effective sterols absorbed by the insect ranged from 0.6 to 6 μg per insect. Competition experiments using different proportions of cholesterol and 24-dihydrolanosterol (from 9:1 to 1:9 mixtures) indicated that abnormal development of H. zea may be induced on less than a 1 to 1 mixture of utilizable (cholesterol) to nonutilizable (24- dihydrolanosterol) sterols. The results demonstrate new structural requirements for sterol utilization and metabolism by insects, particularly with respect to the position of double bonds in the side chain and functionalization in the nucleus. The novel sterol specificities observed in this study appear to be associated with the dual role of sterols as membrane inserts (nonmetabolic) and as precursors to the ecdysteroids (metabolic).