The effect of testicular binding of human chorionic gonadotropin upon the biological activities of the hormone was examined by comparison of the binding and activation properties of 125I-labeled gonadotropin before and after binding to rat testis in vitro. Biologically active 125I-gonadotropin taken up by rat testis was dissocated from testis binding-sites at low pH and evaluated for its ability to bind again to testis, adenylate cyclase activation, and stimulation of steroidogenesis during subsequent incubation with fresh testis. Binding to tissue receptor-sites for 4 hr did not impair the biological properties of gonadotropin, though hormone remaining in the incubation medium had reduced affinity for tissue binding-sites during subsequent incubation with rat testes. In comparison to the original preparation, 125I-labeled gonadotropin previously eluted from specific binding-sites of rat testis showed significantly increased binding activity and stimulation of cyclic AMP and testosterone release during further incubation with rat testes in vitro. The enhancement of biological activity of the eluted hormone is attributable to affinity purification of the original hormone preparation by selective uptake at receptorsites. These results demonstrate that gonadotropin is not inactivated or degraded during combination with gonadotropin receptors of rat testis.