Retinoic acid (RA) inhibits adipocyte differentiation of 3T3-L1 preadipocytes but is effective only early in adipogenesis. RA prevented induction of the adipogenic factors PPARγ and C/EBPα. Using receptor-specific ligands, we determined that the effects of RA were mediated by liganded RA receptors (RARs) rather than retinoid X receptors. Preadipocytes expressed primarily RARα and RARγ; during adipocyte differentiation, RARa gene expression was nearly constant, whereas RARγ1 mRNA and protein levels dramatically decreased. Ectopic expression of RARγ1 extended the period of effectiveness of RA by 24 to 48 h; RARα expression had a similar effect, suggesting functional redundancy of RAR subtypes. Remarkably, RA inhibited differentiation when added after PPARγ1 and PPARγ2 proteins had already been expressed and resulted in the loss of PPARγ proteins from cells. By 72 to 96 h after the induction of differentiation, RA failed to prevent differentiation of even ectopic-RAR-expressing cells. Thus, the unresponsiveness of 3T3-L1 preadipocytes to RA after the induction of differentiation is initially due to the reduction in cellular RAR concentration rather than to the induction of PPARγ. At later times cells continue along the differentiation pathway in a manner which is RA and RAR independent.