Mammalian homologues of the Drosophila Iroquois homeobox gene complex, involved in patterning and regionalization of differentiation, have recently been identified (Mech. Dev., 69 (1997) 169; Dev. Biol., 217 (2000) 266; Dev. Dyn., 218 (2000) 160; Mech. Dev., 91 (2000) 317; Dev. Biol., 224 (2000) 263; Genome Res., 10 (2000) 1453; Mech. Dev., 103 (2001) 193). The six members of the murine family were found to be organized in two cognate clusters of three genes each, Irx1, -2, -4 and Irx3, -5, -6, respectively (Peters et al., 2000). As a basis for further study of their regulation and function we performed a comparative analysis of the genomic organization and of the expression patterns of all six Irx genes. The genes are expressed in highly specific and regionalized patterns of ectoderm, mesoderm and endoderm derived tissues. In most tissues the pattern of expression of the clustered genes, especially of Irx1 and -2 and of Irx3 and -5, respectively, closely resembled each other while those of Irx4 and -6 were very divergent. Interestingly, the expression of cognate genes was found to be mutually exclusive in adjacent and interacting tissues of limb, heart and the laryncho-pharyncheal region. The results indicate that the Irx genes are coordinately regulated at the level of the cluster.