Human cellular nucleic acid binding protein (CNBP) is a zinc finger DNA binding protein of unknown function. The human CNBP cDNA was used as a probe to isolate four structurally distinct but highly homologous mouse liver cDNA clones. Each of the mouse clones exhibited extraordinary sequence conservation with human CNBP cDNA, and the predicted mouse amino acid sequence identities with human CNBP protein ranged from 99 to 100%. Genetic mapping of CNBP genes in interspecific and intersubspecific mouse backcrosses revealed two loci that hybridize to CNBP cDNA at high stringency, located on chromosomes 5 and 6. The subcellular distribution of the CNBP protein was characterized with a specific polyclonal antibody generated against a synthetic peptide from the carboxyl terminus. CNBP was found in the cytosol and the endoplasmic reticulum in subcellular fractions from mouse liver, but was undetectable in nuclear fractions. These data suggest that CNBP is a member of a highly conserved family of cytosolic proteins that may be encoded by multiple dispersed genes.