Aromatase catalyzes the conversion of 4-androstene-3,17-dione to estrogen with the concomitant formation of the minor metabolites 4-androstene-19-hydroxy-3,17-dione(19-hydroxyandrostenedione) and 4-androstene-3,17,19-trione(19-oxoandrostenedione). Microsomes of chinese hamster ovary (CHO) cells expressing human aromatase were isolated to investigate androstenedione metabolism. Relatively greater amounts of the minor metabolites result after limitation of electron flux from NADPH-cytochrome P450 reductase to aromatase. Substitution of NADH for NADPH or limitation of NADPH availability increased minor metabolite formation relative to estrogen formation. Similar changes in metabolite ratios were observed when metabolism was conducted either at high pH (8.3) or in the presence of n-alcohols in the range of 5-200 mM alcohol concentrations. However, conditions of low pH (5.5) or high ionic strength (1 M KCl) resulted in minor changes in metabolite ratios, suggesting little or no effect on electron flux between NADPH-cytochrome P450 reductase and aromatase. Theoretical molar ratios of the resulting metabolites were predicted using a reaction scheme assuming sequential substrate oxidations without reversible intermediate release from the aromatase active site. This model was supported by a close agreement between theoretical and experimental metabolite ratios for a broad range of NADPH concentrations. The results indicate that metabolite ratios provide a sensitive indicator of aromatase-oxidoreductase interactions in the microsomal environment.