The role of second messenger pathways, cyclic AMP, calcium, and protein kinase C (PKC) in the transcriptional regulation of c-fos protooncogene expression in rat Sertoli cells was investigated. c-fos expression was monitored by Northern blot analysis. Although the action of FSH on Sertoli cells is considered to be mediated by cAMP, dibutyryl cAMP (dbcAMP), a potent membrane permeable analog of cAMP, induced much less c-fos mRNA expression than FSH ( < 50%) suggesting that additional cAMP-independent mechanisms may mediate the effect of FSH on c-fos. Specific intracellular inhibitors of PKC decreased c-fos induction in response to FSH by more than 50%. Ionomycin, which increases intracellular free calcium concentration, induced c-fos expression significantly. These data demonstrate that Sertoli cell c-fos mRNA expression is under multifactorial regulation by cAMP, calcium, and PKC.