The positional signaling along the anteroposterior axis of the developing vertebrate limb is provided by the zone of polarizing activity (ZPA) located at the posterior margin. Recently, it was established that the Sonic hedgehog (Shh) mediates ZPA activity. Here we report that a new mouse mutant, Recombination induced mutant 4 (Rim4), and two old mutants, Hemimelic extra toes (Hx) and Extra toes (Xt), exhibit mirror-image duplications of the skeletal pattern of the digits. In situ hybridization of the embryos of these mutants revealed ectopic expression of Shh and fibroblast growth factor-4 (Fgf-4) genes at the anterior margin of limb buds. The new mutation, Rim4, was mapped to chromosome 6 with linkage to HoxAbut segregated from HoxA. No linkage to other known polydactylous mutations was detected. In this mutant, ectopic expression of the Hoxd-11 gene, thought to be downstream of ZPA, was also observed at the anterior margin of the limb buds. All results indicate the presence of an additional ZPA at the anterior margin of limb buds in these mutants. Thus, it appears that multiple endogenous genes regulate the spatial localization of the ZPA in the developing mouse limb bud.