We have characterized the bovine and the human cDNAs encoding the NAP-22 (22 kDa neuronal tissue-enriched acidic protein) homologs. Both bovine and human cDNAs encode proteins of 227 amino acids. The deduced amino acid sequences of the bovine and the human proteins are 63% and 65% identical, respectively, to that of rat NAP-22 protein, strongly suggesting that both the cDNAs characterized encode NAP-22 proteins. They also share 45% and 41% amino acid sequence identities with chicken CAP-23 (23 kDa cytoskeleton associated protein). Several important protein motifs, including myristoylation and phosphorylation sites, are well conserved in sequences and positions in all three mammalian NAP-22 proteins and chicken CAP-23 proteins. The bovine cDNA was characterized further. Southern analysis of the bovine genomic DNA suggests that the bovine NAP-22 protein is encoded by a single-copy gene. RNA blot analysis revealed that the bovine gene for NAP-22 protein encodes a 1.7 kb transcript that is present only in the brain. Our data suggest that the four proteins, bovine and human NAP-22 homologs, rat NAP-22, and chicken CAP-23, have homologous functions in different organisms.