Vitamin A deficiency diminishes Th2-mediated Ab responses, and high-level dietary vitamin A or treatment with the vitamin A metabolite retinoic acid (RA) enhances such responses. To identify a potential mechanism(s) underlying this in vivo activity of vitamin A, we examined the effects of all-trans and 9-cis RA on development of Th1 and Th2 cell populations using in vitro stimulation of Ag-naive ThO cells from the DO11.10 TCR-transgenic mouse. Treatment with 9-cis, but not with all-trans RA, at primary stimulation strongly enhanced Th2 development. IL-4-neutralizing Ab blocked this activity, but IL-12- and IFN-γ-neutralizing Ab did not. Because 9-cis RA regulates gene transcription via either RA receptors or retinoid X receptors (RXRs), we tested the Th2-enhancing activities of the RXR- and RA receptor-selective agonists AGN194204 and 4-((E)-2 -(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl)benzoic acid (TTNPB). AGN194204 strongly enhanced Th2 development, whereas TTNPB did not. This RXR agonist also enhanced Th2 development when purified, naive ThO cells (L-selectinhigh/CD4+) were stimulated with CD3 and CD28 Abs in the absence of APCs. During primary antigenic stimulation of naive ThO cells from DO11.10 mice, AGN194204 increased IL-4 and IL-5 production, decreased IFN-γ production, increased mRNA in responding T cells for genes involved in Th2 development (IL.4, GATA-3, and c-maf), and decreased mRNA for genes involved in Th1 development (IFN-γ, T-bet, and IL-12R). These data show that stimulation of the RXR pathway enhances Th2 development, perhaps by affecting the relative expression of pertinent transcription factors, cytokines, and cytokine receptors.