We report the molecular cloning and characterization of a cDNA derived from a zebrafish gene (ZF-21) related to the mouse homeobox containing gene Hox2.1. Interesting information about the differential conservation of various domains was gained from comparisons between the putative protein sequences from ZF-21 (275 amino acids) and Hox2.1 (279 aa). A separate DNA binding domain including the ZF-21 homeodomain and 36 additional flanking residues is completely identical to the C-terminal part of Hox2.1. As a consequence, these two mouse and zebrafish proteins must have identical DNA binding properties. A lower level of sequence identity between the N-terminal coding regions of ZF-21 and Hox2.1 reduces the total protein homology to 81%. However, short stretches of perfect homology in these N-terminals suggests that the essential biochemical functions are the same. As expected for true homologues, the ZF-21 and Hox2.1 genes also share extensive similarities with respect to non-coding sequences and temporal expression during embryogenesis. The finding of a potential ZF-21 duplication is discussed in relation to functional and evolutionary aspects of vertebrate homeobox genes.