Sertoli cells from rats aged 16 days were cultured in defined medium for 2 days and then treated by addition of fresh medium containing various hormones (treated) or saline (control). The concentration of lactate in the medium was measured as a function of time. The production of lactate measured under these conditions was increased by addition of FSH to the medium. For NIH FSH (13 and 14), ED50 for stimulation of lactate production was approximately 0.05 micrograms/ml. Stimulation was also seen with LH and TSH (ED50, 0.8 micrograms/ml for both hormones). Reasons are given for believing that TSH may possess the inherent capacity to stimulate production of lactate in contrast to LH, which appears to act only by way of contaminating traces of FSH. Dibutyryl cAMP also stimulates lactate production by Sertoli cells. Other hormones tested, including androgens, were without effect. When production of lactate by Sertoli cells was compared with the maximal consumption by spermatids in vitro, it became apparent that Sertoli cells may provide the major source of metabolic substrate for spermatids in the form of lactate. Stimulation of lactate production by FSH was inhibited by puromycin, cycloheximide, and actinomycin D. Evidently this response requires synthesis of new protein and RNA. This effect of FSH may be important in the regulation of spermatogenesis.