The capability of granulosa and theca interna cells, from preovulatory follicles of the domestic hen, to metabolize steroid precursors was evaluated. Granulosa and theca interna cells were isolated from ovarian preovulatory follicles at three different developmental stages: F1, F3 and F5. Tritiated pregnenolone (P5), progesterone (P4), dehydroepiandrosterone (DHEA), androstenedione (A4) and testosterone (T) were employed as precursors and their metabolic products were evaluated. The major metabolite of P5 by granulosa cells was P4, but we also observed low amounts of 5beta-pregnandione. DHEA metabolism by granulosa cells yielded mainly A4, and minute quantities of 5beta-androstan-3,17-dione (5beta-dione) were detected. The only significant metabolite obtained in granulosa cells from A4 was 5beta-dione, whereas T was only transformed into A4. On the other hand, P5 metabolism by theca interna cells yielded A4 as the main product, also P4, 17alpha-OHP4, 17alpha-OHP5, 5beta-pregnandione, and DHEA, were found. When DHEA was the precursor A4 was produced in higher amounts than 5beta-dione. A4 was mainly transformed into 5beta-dione. In similar conditions, T was transformed into A4. These results show that granulosa cells have enzymatic activities of 3beta-hydroxysteroid dehydrogenase/5-4 isomerase (3beta-HSD from P5 and DHEA), 17beta-hydroxysteroid dehydrogenase (17beta-HSD from T) and 5beta-reductase (from P5, DHEA and A4). Whereas theca interna cells have enzymatic activities of cytochrome P450c17 (from P5 and P4), 3beta-HSD (from P5 and DHEA), 17beta-HSD (from T) and 5beta-reductase (from P4, DHEA and A4). These data support the concept that theca interna cells have the ability to synthesize androgens from progestins produced in granulosa cells. In addition, since theca interna cells did not show the capacity to aromatize androgens suggests that interaction between theca interna and theca externa cells occurs in vivo, thus confirming the three cell model for estrogen production. Furthermore, the fact that other metabolites were produced both in granulosa and theca interna cells, but in a different extent, suggests that complex mechanisms are participating in the regulation of steroid synthesis in avian ovary follicles.