A putative messenger RNA (mRNA) sequence, designated C8, that was up-regulated in Sertoli cells prepared from hypophysectomized rats treated with testosterone, was isolated from a Sertoli cell complementary DNA (cDNA) library. The coding region of C8 exhibited 99% identity with rat brain ecto-ATPase and expressed a 60-kilodalton protein following in vitro transcription/translation. Transfection of COS7 cells with C8 cDNA resulted in a marked increase in Ca2+- and Mg2+-dependent ATPase activity in both whole cells and cell homogenates, which is consistent with localization of this enzyme in the plasma membrane. C8 ecto-ATPase steady state mRNA levels were increased within 6 hours and for 3 day, by follicle-stimulating hormone (FSH) in Sertoli cells but not in peritubular cells. In contrast, dibutyryl-cyclic adenosine monophosphate (cAMP) increased ecto-ATPase in both Sertoli and peritubular cells. Testosterone had no significant effect under these conditions. These data indicate that ecto-ATPase mRNA is positively regulated by FSH in Sertoli cells and by cAMP in both Sertoli and peritubular cells. This enzyme may play a role in the control of extracellular signaling by ATP, adenosine, or both in the cells of the seminiferous epithelium.