Retinoids, a group of natural and synthetic analogues of vitamin A (retinol), modulate the differentiation of many cell types. Retinoids are also used for the prevention and treatment of cancer. The actions of retinoids are generally mediated by the retinoic acid receptors (RARs alpha, beta, and gamma) and the retinoid X receptors (RXRs alpha, beta, and gamma). One of the RARs, RARbeta, is expressed at reduced levels in many human carcinomas, and F9 RARbeta(2)(-/-) cells do not growth arrest in response to RA. To determine if RARbeta(2) regulates the expression of a unique set of genes, through the use of subtractive hybridization and DNA array analysis, we have identified and characterized genes that are differentially expressed in F9 RARbeta(2)(-/-) teratocarcinoma cells. These genes, which encode transcription factors, cell surface signal transduction molecules, and metabolic enzymes, include c-myc, FOG1, GATA6, glutamate dehydrogenase, glutathione S-transferase homologue (p28), Foxq1, Hic5, Meis1a, Dab2, midkine, and the PDGF-alpha receptor. These genes are regulated specifically by RARbeta(2) in F9 wild-type (Wt) cells as indicated by their expression profiles in F9 RARbeta(2)(-/-) cells as compared to F9 Wt, RARalpha(-/-), or RARgamma(-/-) cells, and their responsiveness to specific retinoid receptor agonists. The basal expression levels of some of these genes, such as c-myc, are higher in the F9 RARbeta(2)(-/-) cells than in F9 Wt in the absence of exogenous retinoids, suggesting that RARbeta(2) can inhibit gene expression in the absence of a ligand. The RARbeta(2) target genes are transcriptionally activated by retinol, as well as RA, in F9 Wt cells. Because the lack of RARbeta(2) alters both the control of proliferation and differentiation in F9 cells, the genes that we have characterized may mediate key effects of RA, via RARbeta(2), on these processes.