The homeo box, a conserved DNA element first recognized in Drosophila development-controlling genes, is present in the genomes of many higher metazoan species and provides a valuable probe for the isolation of regulatory genes from diverse phylogenetic groups. We have employed these probes to isolate and study the homeo-box genes in sea urchins. As in other species, the sea urchin homeo boxes fall into at least two classes defined by nucleotide sequence similarity to the homeo boxes of the Drosophila Antennapedia (Antp) and engrailed (en) genes. In this study, we characterize the only detectable sea urchin en class homeo box. Its nucleotide sequence similarity and lack of an intron indicate that it is more closely related to the two mouse en class homeo boxes than to the two Drosophila en class homeo boxes. These relationships are most parsimoniously explained if the single sea urchin en class homeo-box gene represents the primitive condition and the two mouse and the two Drosophila en class homeo-box genes represent independent duplications which occurred in the evolutionary lines leading to the vertebrates and arthropods, respectively. The most abundant en class gene transcripts detected by gel transfer analysis of RNA extracted from sea urchin tissues were found in Aristotle's lantern. Rare transcripts were present in ovary, testis and coelomocytes.