Sertoli cell monolayers were prepared from 19-day-old rat testes. On day 7 of culture cells were incubated for 24 hr in the presence or absence of ovine follicle stimulating hormone (oFSH). Cells were harvested, and adenylyl cyclase responses of the membrane particles to FSH, human chorionic gonadotropin (hCG), isoproterenol, and fluoride (F-) were examined in the presence of either GTP or the nonhydrolyzable guanylyl nucleotide GMP-P(NH)P. Culturing the cells in presence of FSH caused a hormone specific desensitization of FSH-responsive adenylyl cyclase, whereas responses to isoproterenol and fluoride were unaffected. Activation of Sertoli cell adenylyl cyclase by GTP and GMP-P(NH)P showed no difference between cells preincubated with or without FSH, indicating that FSH did not change the activity of the G/F (or N) component or its interaction with the catalytic subunit of the adenylyl cyclase. FSH-responsive adenylyl cyclase in cultured Sertoli cells has been shown to be selectively desensitized by homologous hormone. The mechanism may involve alteration or loss of the FSH receptor or changes in the "coupling" of the FSH receptors to the G/F component of the adenylyl cyclase, since there was no alteration in the guanylyl nucleotide and fluoride activation.