The RARγ gene generates two major isoforms, RARγ1 and RARγ2, which originate from two distinct promoters. We report here the engineering of mice lacking RARγ1, but in which RARγ2 is normally expressed. The effect of this null mutation has been compared with those previously described for RARγ2 and all RARγ isoforms (total RARγ gene inactivation), both in single mutants and in double mutants bearing additional null mutations in their RARα, RARβ or RXRα genes. RARγ1 mutants, but not RARγ2 mutants, displayed a subset of the abnormalities exhibited by total RARγ null mutants (growth deficiency, abnormal cricoid cartilage and occasional cervical vertebra defects), suggesting that RARγ1 is the main isoform mediating the corresponding RARγ functions. Interestingly, cricoid cartilage defects were also found in a fraction of heterozygote animals for the RARγ1, RARγ or RARα mutations, indicating that wild type levels of RARs are required for the normal morphogenesis of this structure. Compound RARα/RARγ1 and RARα/RARγ2 double null mutants exhibited only a small fraction of the defects found in RARα/RARγ double null mutants. Moreover, these defects were often partially penetrant, or corresponded to a less severe form. However, they occurred preferentially in certain compound mutants, demonstrating that given isoforms mediate specific functions of RARγ in the context of a RARα null background. In a RXRα null background, both RARγ1 and γ2 isoform mutations resulted in increased severity of the RXRα null ocular phenotype. Together, the present observations indicate that the functions of the two RARγ isoforms overlap to a large extent, but also that each of these isoforms exhibits a limited functional specificity. Furthermore, the occurrence of morphological defects in heterozygote mutants for a single RAR isoform provides a basis for explaining the strong conservation of these isoforms during vertebrate evolution.