The impact of compartmental expression of steroidogenic enzymes and of changes in flux through delta5 and delta4 metabolism on sex steroid synthesis was investigated by rebuilding pathways using recombinant enzyme expression by infection of insect cells with recombinant baculovirus constructs . Human cytochromes 17alpha-hydroxylase/17,20-lyase (P450c17) and aromatase (P450arom), always coexpressed with their redox partner NADPH-P450 oxidoreductase (CPR) and 3beta-hydroxysteroid dehydrogenase/delta5-4 isomerase (3betaHSD; types 1 or 2), were compartmentally expressed in different cell populations or coexpressed together with pregnenolone (100 nM) as substrate. Estrone was compared among cell compartments expressing different enzyme combinations or in cells coexpressing all enzymes (experiment 1). Additionally, P450c17, 3betaHSD, and CPR were all coexpressed, and androstenedione was measured in cells with different 3betaHSD expression levels or activity using an inhibitor, trilostane (experiment 2). Steroids were measured by immunoassay and mass spectrometry. In experiment 1, partitioning of P450c17, P450arom, and 3betaHSD markedly decreased estrone synthesis in comparison to cells coexpressing enzymes in different combinations. However, partitioning P450arom with 3betaHSD from P450c17 in different cell populations resulted in more estrone than either of the other two-cell compartment models. In experiment 2 (cells coexpressing P450c17, 3betaHSD, and CPR), androstenedione secretion was (paradoxically) higher at lower levels of 3betaHSD, and partial inhibition of 3betaHSD by trilostane also increased androstenedione when 3betaHSD expression was high. We conclude 1) that tissue or cell-specific, partitioned expression of sex steroid synthesizing enzymes limits rather than maximizes estrogen synthesis and 2) that limiting metabolism by 3betaHSD can paradoxically promote androgen synthesis when 3betaHSD expression is high by promoting delta5-steroid flux.