The in vitro response of Sertoli cells isolated from adult rat testes to testosterone (T) and follicle-stimulating hormone (FSH) treatment was investigated. Sertoli cells from >70-day-old Sprague-Dawley rats were isolated by a combined enzymatic treatment followed by the removal of the majority of contaminating germ cells with immobilized peanut agglutinin lectin. Sertoli cells were then cultured for 6-10 days, forming a confluent layer with a cell viability of >83% and 74-77% purity. The contaminating cells were peritubular cells (4-6%), pachytene spermatocytes (4-5%), round spermatids (<2%), elongated spermatids (<1%), and degenerating germ cells (14.8%). The proportion of degenerating germ cells decreased from 14.8% to 8.6% between days 6 and 10 in culture. After a prestimulation culture period of 4 days, FSH treatment over a 2-day period resulted in a dose-related increase of inhibin with a median effective dose (ED50) value of 36.7+/-20.4 ng/ml in comparison with an ED50 value of 4.4+/-0.9 ng/ml obtained with immature Sertoli cell cultures from 20-day-old rats. Mature Sertoli cells, in contrast to immature Sertoli cells, were unresponsive to combined FSH + T treatment for the production of the cell adhesion protein N-cadherin. FSH treatment promoted the in vitro binding of round spermatids isolated from adult testis to adult Sertoli cells in coculture. It is concluded that mature Sertoli cells in culture are responsive to FSH in terms of inhibin production and round-spermatid binding. The lack of an FSH + T-induced increase in N-cadherin or round spermatid binding is attributed to either a reduced sensitivity, or an alteration in the regulation of mature Sertoli cells by FSH + T.