Earlier studies showed that the predominant steroid (estradiol E2, testosterone T, progesterone P4) secreted by in vitro cultured amphibian (Rana nigromaculata) ovarian follicles varied with their size. E2 was mainly produced by medium-sized follicles, T by intermediate-sized follicles, and P4 by the largest follicles. Experiments were carried out to ascertain whether the activities of steroidogenic enzymes changed during follicle development and were responsive to gonadotropic stimulation. Enzyme activities were measured indirectly by monitoring conversion of exogenously added substrates to products during in vitro culture of isolated follicles. Different stage follicles were cultured in the presence or absence of frog pituitary homogenate (FPH, 0.1 pituitary/2 ml) and/or various steroid precursors (25-200 ng/2 ml). Amounts of E2, T, androstenedione (AD), 17 alpha-hydroxyprogesterone (17 alpha-OHP4), or P4 secreted into the medium were measured by RIA. Exogenous pregnenolone (P5) was converted to P4 by all types of follicles in a dose-dependent manner in the absence of FPH. Addition of FPH markedly enhanced medium levels of P4 in all sized follicles. Highest levels of P4 were presented in cultures containing the largest follicles. Such follicles were much less efficient than intermediate follicles in metabolizing P4 to AD or T. FPH suppressed conversion of exogenous 17 alpha-OHP4 but not androstenedione to testosterone by the largest follicles. Exogenous T was converted to E2 only by medium-sized follicles and FPH had little or no stimulating or inhibiting effect on this process in either medium- or intermediate-sized follicles.