Sertoli cells were isolated from the testes of 3-week-old sterile rats (prenatally irradiated) and incubated for 3 days in the absence of added hormones. Subsequently the effects of follitropin and insulin on glucose metabolism were investigated using this in vitro system. A marked stimulation of net lactate production by either follitropin or insulin was observed within 3 h after addition of the hormones. This response was not inhibited in the presence of the protein synthesis inhibitor cycloheximide. Production of cAMP by the Sertoli cells was markedly enhanced by follitropin, but not at all by insulin. The addition of 0.5 mM dibutyryl cAMP to the incubation medium also resulted in a rapid increase of the rate of lactate production by the Sertoli cells. The stimulation of lactate production by follitropin and insulin was dose-dependent (ED50 of approx. 10 ng NIH-FSH-S13/ml and of approx. 50 ng insulin/ml). It is suggested that the observed effects of insulin on Sertoli cells are mediated via insulin receptors, rather than via receptors for insulin-like growth factors. Within 18 h after addition of either follitropin or insulin the cells became refractory with respect to lactate production to the homologous hormone, whereas the cells could still respond to the heterologous hormone. It is concluded, that follitropin and insulin, acting via different mechanisms, exert similar rapid stimulatory effects on glucose metabolism by Sertoli cells from immature rats in vitro. These effects are not dependent on de novo protein synthesis and may differ from long-term trophic effects of follitropin, insulin, and/or insulin-like growth factors.