The animal-vegetal (A-V) axis of sea urchin embryos is morphologically evident at the 16-cell stage of development. Mesomeres, macromeres, and micromeres are arrayed along the A-V axis. The vegetal micromere differentiates into the skeletogenic mesenchyme and functions as a signaling center. To date, no zygotic or maternally specified gene with restricted expression in the micromere at the 16-cell stage has been reported. We performed subtraction PCR and dot blot hybridization using poly(A)+ RNA extracted from the micromere (tester) and the mesomere (driver) in order to identify micromere-specific genes. Using a cDNA fragment identified in this screen, we isolated four similar but distinct cDNA clones from a library, which corresponded to a group of genes that we refer to as the micro1 family. The micro1 family encoded putative transcription factors with a homeodomain which had 87-95% identity between family members. The most highly conserved protein was encoded by PlHbox12 from Paracentrotus lividus (71-76% identity among family members). Northern blot hybridization and in situ hybridization demonstrated that micro1 was transiently activated during the early cleavage stages and that the transcript was restricted to the micromere. Thus, the expression domain was complementary to that of PlHbox12 along the A-V axis. The micro1 gene family has at least six loci, including polymorphic alleles, which are probably clustered in the genome. PlHbox12 and micro1 constitute a novel family of paired-like class homeobox genes. Phylogenetic analyses suggest that PlHbox12/micro1 evolved exceptionally rapidly.