The secretion of plasminogen activator (PA) has been found to be highly stage-specific during rat spermatogenesis. It is maximal in Stages VII and VIII of the cycle. At these stages, seminiferous tubules contain primitive type A1 spermatogonia, preleptotene and midpachytene primary spermatocytes, round and maturation-phase spermatids and Sertoli cells. The last cell types are the most likely sources of PA. To investigate which cell type might be involved in the regulation of PA secretion, we have sequentially isolated 1-mm segments of rat seminiferous tubules from Stages VI-IX with transillumination-assisted microdissection and measured PA secretion using 125I-labeled fibrinogen as substrate. In another experiment, spermatogenia were killed by 300 rads of x-rays and PA secretion was analyzed during the absence of desired germ cell classes. The results support the idea that upon their detachment from the basal lamina, preleptotene-stage primary spermatocytes have the major stimulatory action on the PA secretion of the seminiferous tubules. Other phenomena such as spermiation, phagocytosis of residual bodies or opening up of the Sertoli cell junctions seem to influence PA secretion to a lesser extent.