The testicular Sertoli cells support spermatogenesis by providing a microenvironment and structural support for the developing germ cells. Sertoli cell functions are regulated by the gonadotropin FSH. Sertoli cells become a terminally differentiated nongrowing cell population in the adult. In response to FSH, the Sertoli cells express a large number of differentiated gene products, such as transferrin, which transports iron to the developing germ cells. Previously, members of the basic helix-loop-helix (bHLH) family of transcription factors have been shown to influence FSH-mediated gene expression in Sertoli cells. The functions of the bHLH proteins are modulated by Id (inhibitor of differentiation) proteins, which lack the DNA-binding basic domain. The Id proteins form transcriptionally inactive dimers with bHLH proteins and thus regulate cell proliferation and differentiation. The current study investigated the expression and function of Id proteins in the postmitotic Sertoli cell. Freshly isolated and cultured Sertoli cells coexpress all four isoforms of Id (Id1, Id2, Id3, and Id4), as determined by immunoprecipitation with isoform-specific anti-Id antibodies, RT-PCR, and Northern blot analysis. Id2 and Id3 expression levels seem higher than Id1. Interestingly, the expression of Id4 in Sertoli cells is only detectable after stimulation with FSH or cAMP. The Id1 expression is down-regulated by FSH and cAMP, whereas Id2 and Id3 levels remain unchanged in response to FSH. In contrast, serum induces the expression of Id1, Id2, and Id3. Treatment of Sertoli cells with serum significantly reduces the expression of the larger 4-kb Id4 transcript and promotes the presence of a novel 1.3-kb transcript of Id4. The regulatory role of FSH in the expression of all four isoforms of Id is mimicked by a cAMP analog, suggesting that the actions of FSH are mediated through the protein kinase A pathway. An antisense approach was used to study the functional significance of Id proteins in Sertoli cells. Antisense to Id1 stimulated transferrin promoter activity in a transient transfection assay. Interestingly, an antisense to Id2 down-regulated transferrin promoter activity. Id3 and Id4 antisense oligonucleotides had no effect on FSH-mediated transferrin promoter activation. Contrary to the hypothesis that Id proteins have redundant functions, the results of the current study suggest that Id1, Id2, Id3, and Id4 are differentially regulated and may have distinct functions. Id1 may act to maintain Sertoli cell growth potential, whereas Id2 and Id4 may be involved in the differentiation and hormone regulation of Sertoli cells.