We investigated whether insulin plays a role in the proliferation and androgen production of chick testis cells. Testes from 18-d-old chick embryos or newly hatched chickens were dissociated and precultured in the presence of fetal bovine serum (FBS) for 24 h. After this period, testis cells from 18-d-old chick embryos were cultured in serum-free medium for 1 h with 0, 10, 50, or 100 microg/mL of insulin and were then exposed to human chorionic gonadotropin (hCG) for 3 h. In addition, some cells were incubated for 18 h with only insulin or insulin plus hCG. Androgens were measured by radioimmunoassay in the spent media. To study the influence of insulin on testis cell proliferation, cells were exposed to insulin for 18 h. A pulse of 3H-thymidine was added thereafter. We found that 18-d-old embryonic testis cells responded to hCG, increasing androgen production. Incubation with insulin for 1 h did not affect basal androgen production but modified the subsequent response to hCG. The addition of insulin plus hCG for 18 h resulted in important downregulation of the hCG effect. In addition, insulin significantly increased the proliferation of embryonic testis cells. The cells from testes of newly hatched chickens were precultured as described for embryonic cells and then exposed to insulin for 1 h in a serum-free medium. This treatment significantly increased the basal androgen production. Insulin also significantly enhanced the response to hCG of the testis cells from newly hatched chickens. These results strongly suggest that insulin has a role in the activity and in the proliferation of cultured testis cells throughout the perinatal period.