Sertoli cells, the somatic epithelial cells of the seminiferous tubules, provide both structural and biochemical support for developing male germ cells. The Sertoli cells are targets of retinoid action in the testis. We have found that FSH, (Bu)_2cAMP, and leukemia inhibitory factor elicit substantial changes in the metabolism of [^3H]retinol (vitamin A) in primary cultures of purified rat Sertoli cells. Addition of (Bu)_2cAMP for 2 h or FSH for 6 h results in a 3-fold increase in the metabolism of [^3H]retinol to [^3H]retinoic acid ([^3H]RA); the esterification of [^3H]retinol to [^3H]retinyl esters, especially [^3H]retinyl palmitate, is also increased by approximately 5-fold. The addition of 1 µM all-trans-RA also elicits changes in [^3H]retinol metabolism, but in this case the metabolism of [^3H]retinol to [^3H]RA is inhibited, whereas the metabolism of [^3H]retinol to [^3H]retinyl esters is increased by over 50-fold. Leukemia inhibitory factor increases the esterification of [^3H]retinol by 2- to 3-fold. FSH leads to a reduction in the level of cellular retinol binding protein I transcripts, whereas RA increases the cellular retinol binding protein I messenger RNA level by about 2-fold at approximately 24 h. Levels of AHD-2 (aldehyde dehydrogenase-2) and RALDH-2 (retinal dehydedehydrogenase-2) messenger RNAs, which encode enzymes that convert [^3H]retinaldehyde to [^3H]RA, are increased by about 2-fold by FSH, whereas no change in CYP26 (RA hydroxylase) expressionis seen. Our results suggest that one function of FSH (and/or (Bu)_2cAMP) in Sertoli cells is to increase the metabolism of retinol to the biologically active metabolite RA and to retinyl esters.