Using the polymerase chain reaction with primers corresponding to conserved regions in the kinase domain of protein-tyrosine kinases, we amplified segments of several protein-tyrosine kinase genes from Hydra vulgaris, a member of the ancient metazoan phylum Cnidaria. Characterization of cDNA clones for one of these genes, HTK16, revealed that it encodes a non-receptor protein-tyrosine kinase with two SH2 domains but no SH3 domain. In this regard the predicted HTK16 protein resembles two mammalian non-receptor protein-tyrosine kinases, the products of the ZAP-70 and syk genes. However, the HTK16 protein contains five ankyrin-like repeats, a structural motif which has not previously been found in protein-tyrosine kinases. The HTK16 protein also contains a potential tyrosine phosphorylation site in its carboxyl-terminal tail which resembles the phosphorylation site in members of the src family. RNA hybridization analysis indicates that the HTK16 gene is expressed in epithelial cells, cells which also express the Hydra homologue of the src protein. Our finding of the HTK16 gene in Hydra indicates that diversification of genes encoding non-receptor protein-tyrosine kinases was a very early event in metazoan evolution.