We have found that follicle stimulating hormone (FSH) stimulates the formation of colonies by 10 day old rat testicular cells in primary culture. With the ability to study hormonal actions on cells in culture, the problems of hormonal interacctions seen in in vivo experiments are overcome. We, therefore, propose to 1) investigate the hormone specificity of colony formation by immature rat testis cells and investigate the cellular processes involved in the response and 2) purify the FSH-responsive cells and investigate their steroidogenic capabilities in culture. In addition, we have purified two germinal cell populations (spermatogonia and preleptotene spermatocytes) from the immature rat testis. To test the roles of hormonal and cellular interactions in the differentiaton of these important germinal cells, pure germinal cells will be cocultured with FSH-responsive cells. Techniques have been developed for quantitation of cell maintenance and differentiation of attached and non-attached testicular cells in culture. The application of these criteria to cultures of purified cells treated with known hormones offers an experimental situation where, for the first time, specific hormonal and cellular interactions involved in spermatogenesis can be studied. We can, therefore, determine the cell type responsive to FSH and elucidate the metabolic responses involved in the FSH stimulation, determine if interactions with other hormones affect the FSH response, and determine if these hormonal or cellular interactions play a role in the differentiation of early germinal cells.