CPI-17 is a phosphorylation-dependent inhibitor of myosin phosphatase. cDNA clones encoding CPI-17 were isolated from a human aorta library. Overlapping clones indicated two isoforms: CPI-17 alpha was 147 residues and mass of 16.7 kDa; CPI-17 beta (120 residues, mass 13.5 kDa) resulted from a deletion in the a-isoform of 27 residues, sequence 68-94. N-terminal 67 residues of all CPI-17 isoforms (human, porcine, rat and mouse) were highly conserved (for the human and porcine isoforms the identity was 91%). The presence of the two human isoforms was detected from cDNA sequences amplified by RT-PCR and by Western blots on human aorta. The cloned human CPI-17 gene indicated 4 coding exons and CPI-17 beta was an alternative splice variant due to deletion of the second exon. FISH analysis located the human CPI-17 gene on chromosome 19q13.1.