Vertebrates are highly sensitive to both retinoic acid (RA) deficiency and excess. The RA signal is thought to be transduced by nuclear receptors (the RAR and RXR families) which activate the expression of target genes via cis-acting transcriptional enhancer elements. Each of the three RAR genes, RAR alpha, RAR beta, and RAR gamma, gives rise to several isoforms by differential usage of two promoters and alternative splicing. RAR beta 2 is the most abundant of the four RAR beta isoforms, and its transcription is spatially and temporally restricted in developing embryos, suggesting that it might perform specific functions. Furthermore, RAR beta 2 expression can be induced via a retinoic acid response element located in its promoter region. This RA effect is particularly interesting since under conditions of RA excess, RAR beta 2 promoter activity and transcript accumulation are induced in regions of developing embryos in which malformations subsequently appear, such as the craniofacial region, the hindbrain, and the limbs. These findings have led to the suggestion that the RAR beta 2 isoform might mediate some of the teratogenic effects of RA. In this study, we have eliminated RAR beta 2 expression by targeted gene disruption. RAR beta 2 null mutants exhibit an apparently normal phenotype, indicating that other RARs must compensate for RAR beta 2 sufficiently well to allow normal prenatal and postnatal development to proceed. By challenging RAR beta 2 null embryos with teratogenic doses of RA, we have also directly addressed the question of whether RAR beta 2 is required for mediating RA-induced malformations.