We report here that in three preaxial polydactylous mutants in the mouse, namely, 1st, 1x, and Xp1, ectopic expression of the Shh and Fgf-4 genes can be detected at the anterior margin of limb buds. These and three other mutants, namely, Rim4, Hx, and Xt1, which we described in our previous study, all appeared to form a duplicated zone of polarizing activity (ZPA) at the anterior margin of the limb bud. We studied the spatial and temporal pattern of expression of the Gli3 gene, which is affected in a loss-of-function type of mutation, Xt1. The expression domain of Gli3 appeared to be complementary to the ZPA region and the gene was expressed prior to Shh. The results support the hypothesis that GLI3 functions in the anterior portion of limb mesoderm to suppress the expression of Shh. In Drosophila, the gene ci, the fly homologue of Gli, functions to repress hh, suggesting that the negative regulation of the expression of hedgehog by genes belonging to the GLI-kruppel family has been conserved from flies to mice. Finally, we found that the polydactylous phenotype of the mutants Rim4, Xt, 1st, and 1x could be abrogated by the crossing with an inbred strain derived from wild mouse, MSM, whereas the phenotype of Xp1 could not. These results indicate the presence of a modifier gene(s) that can influence the mutant phenotype and also that the mutations could be classified into two categories with regard to the mode of interaction with the modifier gene(s). Thus, this study revealed a multigenic control in the establishment of the anteroposterior axis in mouse limb development.