Changes in rat and human testicular human chorionic gonadotropin (hCG) binding sites induced by hCG were estimated in vivo and in vitro. After a single administration of hCG, the specific hCG binding sites were significantly reduced for 7 and 5 days in rat and human testes, respectively. However, hCG binding sites had recovered to pretreatment values by the 14th day after the administration. Occupied hCG binding sites measured in both rat and human testes accounted for about half of the reduced binding sites on the day after administration of hCG, but thereafter the number of sites occupied was not correlated with the reduction of the binding induced a dose-related significant loss of the specific hCG binding sites for 7 and 5 days in rat and human testes, respectively. Thereafter, the number of binding sites gradually increased. These patterns of change in hCG binding sites in vitro were similar to those in vivo. These findings suggest that the reduction in hCG binding sites in rat and human testes by hCG is due not only to occupancy but also down-regulation of the binding sites. In conclusion, the present testicular organ culture method is useful to study hormonal regulation of testicular function, especially in human testes.