We demonstrate that androgens rapidly and specifically increase intracellular calcium in Sertoli cells, investigate the mechanism, and suggest the unifying hypothesis that calcium might be a common intracellular molecular effector to explain the known synergism between FSH and testosterone (T) action on Sertoli cells in support of spermatogenesis. In freshly isolated Sertoli cells, T and its 5 alpha-reduced metabolite dihydrotestosterone increased intracellular calcium from 83 +/- 4 to 147 +/- 8 and 167 +/- 29 nM, respectively, whereas estradiol had minor (117 +/- 9 nM) and progesterone no (80 +/- 6 nM) effect. The effect of T was rapid (20-40 sec) and inhibited by 1) preincubation with either a pure nonsteroidal antiandrogen (hydroxyflutamide) or a 5 alpha-reductase inhibitor (finasteride) or 2) removal of extracellular calcium (47 +/- 4 nM) or pharmacological blockade of voltage-activated (62 +/- 5 nM) or voltage-independent (55 +/- 14 nM) membrane calcium channels. These findings suggest that the T-induced rise in Sertoli cell cytosolic calcium involves sequential 5 alpha-reduction, binding to a classical androgen receptor, and activation of transmembrane influx of extracellular calcium. Immobilization of T by conjugation to a large carrier molecule (BSA) to prevent steroid entry into Sertoli cells also resulted in a rapid increase in cytosolic calcium to a similar magnitude as unconjugated T, consistent with a plasma membrane site of action. This finding together with the rapid cytosolic calcium rise caused by T argues for the possible existence of a short term, nongenomic effects in hormonal regulation of Sertoli cell function in addition to the well known, slower genomic response.