Thermophilic (45-75°C) iron-reducing bacteria obtained from two sedimentary basins in Virginia and Colorado, USA, reduced amorphous Fe(III) oxyhydroxide to form magnetite-rich (>60% in most samples) iron oxides in acetate- or H2/CO2-enriched cultures. The mineralogical compositions of the iron oxides were determined by X-ray diffraction and oxidation state analyses. Significantly lower Eh values (<-300 mV) occurred in the enrichment cultures than in the abiotic controls (Eh > -100 mV). The pH values in acetate-enriched cultures did not change significantly from the starting value (∼7.2); however, pH values as high as 8.7 were found in the H2/CO2-enriched cultures when abundant siderite was formed in addition to magnetite. The microbial production of magnetite and siderite was consistent, on a thermodynamic basis, with Eh-pH conditions determined for these experiments. Examination of the magnetite-rich iron oxides by scanning electron microscopy showed extracellular aggregates of <200 nm and no distinguishable increase in particle size over a period of 20 days. Average values of oxygen isotope fractionation between the magnetite-rich iron oxides (io) and water (wt), expressed as 103 ln αio-wt, ranged from -0.09‰ at 50°C to -1.08‰ at 70°C. These values did not differ significantly among various cultures of different growth rates, suggesting that a kinetic isotopic effect is either unimportant or reproducible during microbial magnetite formation. Results of this research indicate that studies combining microbial activity, solution chemistry, mineralogy, and oxygen isotopes can provide insight into the environmental conditions and mechanisms for biogenic iron mineral formation in natural systems.