We have developed a rapid and convenient method of Sertoli cell preparation for studying the growth kinetics of these cells in in vitro culture. Datura Stramonium agglutinin (DSA)-coated dishes were used to rapidly purify single Sertoli cells from immature rat testis. We have monitored by immunohistochemical markers the degree of contamination of our Sertoli cell preparation by other cell types. The cell preparation is essentially free of germ cells and interstitial cells and contains a minimal percentage of myoid cells. Sertoli cells isolated with this method retain functional activities such as the FSH responsiveness in terms of cAMP production. In addition, we have studied the proliferative activity of Sertoli cells isolated by lectin binding from rats of different ages. Sertoli cells exhibited a characteristic pattern of proliferation which was a function of the donor animal age. The proliferative activity of isolated Sertoli cells decreased with age, being much higher in 3 day-old rats than in older animals. A similar pattern was observed when the mitotic activity of Sertoli cells in response to mitogens present in the testicular extracts from 5 day-old rats was evaluated. The method described here reduces or eliminates many of the drawbacks of the conventional procedures used to isolate Sertoli cells, thus providing a useful tool in studies of growth kinetics and regulation of cell proliferation in vitro.