Leydig cells were isolated from the perch testes belonging to the pre-spawning stage by collagenase treatment and mechanical separation followed by percoll gradient. They were incubated in vitro either for 5 h or at different times in the absence (control) or presence of piscine gonadotropin (GTH, 2 microg (1 x 10(6) cells)(-1)) or 3,5,3'-triiodothyronine (T3, 50 ng (1 x 10(6) cells(-1)) or T3-induced protein (TIP, 2 microg (1 x 10(6) cells)(-1)). 3Beta-hydroxysteroid dehydrogenase/delta5-delta4-isomerase (3beta-HSD) activity was determined by the conversion of [3H]delta5-dehydroepiandrosterone (DHEA) to [3H]delta4-androstenedione or [3H]delta5-pregnenolone to [3H]delta4-progesterone (P4) or by spectrophotometric estimation of NADH formation from NAD. T3 significantly increased (P < 0.01) both delta5-DHEA to delta4-androstenedione and delta5-pregnenolone to delta4-P4 conversion in Leydig cells indicating stimulation of 3beta-HSD activity. T3 stimulation of 3beta-HSD activity could be inhibited by cycloheximide (50 microg ml(-1)) suggesting the involvement of T3-induced protein (TIP) which was isolated and purified earlier in this laboratory from goat Leydig cells . Addition of TIP or GTH significantly stimulated Leydig cell 3beta-HSD activity (P < 0.01). However, there was a difference between TIP and GTH stimulation in time kinetic study where TIP enhanced 3beta-HSD activity at 1 h (P < 0.05), reached its peak at 3 h (P < 0.01) and then plateaued till 8 h. GTH, on the other hand, did not show any stimulation of 3beta-HSD activity for 2 h, stimulation was marked only at 3 h (P < 0.05), reached a peak at 6 h (P < 0.01) and then leveled off. Determination of Km and Vmax of the enzyme showed an increase in the velocity of reaction by GTH with unaltered Km. TIP increased both velocity and affinity of the enzyme. GTH significantly increased the synthesis of 3beta-HSD protein at 3 h (P < 0.01) reaching maximal stimulation at 6 h which clearly coincided with the enzyme activity. In contrast, TIP had no effect on 3beta-HSD protein synthesis, but its direct addition to 3beta-HSD enzyme preparation in vitro caused significant augmentation of the enzyme activity (P < 0.01) suggesting thereby its modulatory effect on the enzyme. Results, therefore, show that although both T3 and GTH stimulated perch testicular Leydig cell 3beta-HSD activity, T3 effect was not direct but mediated via TIP and there is a clear distinction between GTH and TIP stimulation. GTH increased the enzyme activity by stimulating 3beta-HSD protein synthesis while TIP acts directly on the enzyme modulating it from less active to more active state.