BACKGROUND: Derivatives of retinol (vitamin A), commonly referred to as retinoids, signal through retinoic acid and retinoid X receptors (RARs/RXRs) and are essential for normal limb formation. Retinoid imbalances or perturbations in receptor function result in aberrant limb development. To examine the mechanisms underlying retinol-induced limb defects, we determined the responsiveness of limbs from RAR␣1 Ϫ/Ϫ ␥ mice to excess retinol in vitro. METHODS: RAR␣1 Ϫ/Ϫ ␥ ϩ/Ϫ mice were bred and their embryos were recovered at gestational day (GD) 12.5. The forelimbs were excised and cultured in vitro in the presence of all-trans retinol acetate (0, 1.25, 12.5, or 62.5 M) for 6 days. The expression profiles of genes known to affect chondrogenesis (sox9 and col2a1) and limb outgrowth (meis1, meis2, and pbx1a) were examined by real-time qRT-PCR following retinol exposure for 3 hr. RESULTS: Whereas RAR␣1 Ϫ/Ϫ ␥ ϩ/ϩ and RAR␣1 Ϫ/Ϫ ␥ ϩ/Ϫ limbs exhibited deleterious effects on limb outgrowth and chondrogenesis in the presence of exogenous retinol, this outcome was significantly attenuated in RAR␣1 Ϫ/Ϫ ␥ Ϫ/Ϫ limbs. The expressions of sox9 and col2a1 were significantly decreased in retinol-exposed RAR␣1 Ϫ/Ϫ ␥ ϩ/ϩ limbs. In contrast, expression was not altered in limbs from their RAR␣1 Ϫ/Ϫ ␥ ϩ/Ϫ or RAR␣1 Ϫ/Ϫ ␥ Ϫ/Ϫ littermates. Retinol exposure upregulated the expression of meis1 and meis2 in RAR␣1 Ϫ/Ϫ ␥ ϩ/ϩ limbs; however, in RAR␣1 Ϫ/Ϫ ␥ Ϫ/Ϫ limbs the expression of both genes was unresponsive to retinol. Pbx1a remained unresponsive to retinol treatment in all genotypes. CONCLUSION: In the absence of RAR␣1, RAR␥ is a functionally important mediator of retinoid-induced limb dysmorphogenesis. Birth Defects Research (Part A) 76: 39 -45, 2006.