The procession of round spermatids through stages VII and VIII of the rat spermatogenic cycle is critically dependent on testosterone (T). When intratesticular T levels are reduced, round spermatids appear to slough from the seminiferous epithelium, resulting in the disappearance of elongated spermatids. We hypothesized that T-dependent cell adhesion molecules are involved in Sertoli cell-round spermatid interactions. This study examined the hormonal regulation of one candidate cell adhesion molecule, N-cadherin, in vitro and its participation in Sertoli cell-round spermatid adhesion in coculture. Sertoli cells were isolated from 20-day-old Sprague-Dawley rats; treated with FSH and T, alone or in combination; and incubated for 48 h before determination of N-cadherin concentrations in Sertoli cell extracts by RIA. Both FSH and T significantly increased the cellular content of N- cadherin (3.7-fold), whereas FSH or T alone had no effect. Round spermatids were isolated from adult rats, and their adhesion to Sertoli cells was assessed in a 48-h coculture in the presence of FSH, T, or FSH plus T. Adherent round spermatids were quantitated by histological evaluation after staining with the periodic acid-Schiff reaction. A dose-dependent increase in round spermatid density (number of round spermatids bound per 10,000-μm2 Sertoli cell culture surface area) was observed with increasing T doses (7- 28 ng/ml) in the presence of FSH (1 μg/ml), whereas FSH and T alone at these doses produced no effect. T also increased the N-cadherin content of the cocultures in a dose-dependent manner in the presence of FSH. Addition of an N-cadherin antiserum to the Sertoli cell-round spermatid coculture in the presence of FSH and T significantly (P <0.0001) reduced round spermatid density by 65%. It is concluded that both the production of N-cadherin by Sertoli cells and the binding of round spermatids to Sertoli cells are stimulated in a synergistic manner by T and FSH. Furthermore, the immunoneutralization data suggest the active involvement of N-cadherin in round spermatid-Sertoli cell adhesion in vitro. N-Cadherin may be one of the factors that subserve the androgen-dependent process of round to elongated spermatid maturation.