In order to test current hypotheses on the early evolution of Antp-class (HOM/Hox) genes in metazoan animals, we surveyed the diploblastic hydrozoan Eleutheria dichotoma for the presence of these genes. By means of PCR with different sets of degenerate primers, three new homeobox sequences were identified, in addition to two previously found fragments. Following RACE amplification of the 3' and 5' flanking regions, we classified all five genes, Cnox-1 to Cnox-5, as belonging to the Antp-class. Homeodomain identity to the most similar HOM/Hox genes from Drosophila and human ranges from 58 to 72%. Each of the Cnox genes contains diagnostic residues of more than one Antp-class family known from triploblastic animals. These data are at odds with a previously proposed ancestral HOM/Hox cluster composition, with respect to both the number and types of Antp-class homeobox genes found. (C) 1996 Academic Press, Inc.