Attempts to understand the physiological roles of the protein kinase inhibitor (PKI) proteins have been hampered by a lack of knowledge concerning the molecular heterogeneity of the PKI family. The PKI gamma cDNA sequence determined here predicted an open reading frame of 75 amino acids, showing 35% identity to PKI alpha and 30% identity to PKI beta 1. Residues important for the high affinity of PKI alpha and PKI beta 1 as well as nuclear export of the catalytic (C) subunit of cAMP-dependent protein kinase were found to be conserved in PKI gamma. Northern blot analysis showed that a 1.3-kilobase PKI gamma message is widely expressed, with highest levels in heart, skeletal muscle, and testis. RNase protection analysis revealed that in most tissues examined PKI gamma is expressed at levels equal to or higher than the other known PKI isoforms and that in several mouse-derived cell lines, PKI gamma is the predominant PKI message. Partial purification of PKI activities from mouse heart by DEAE ion exchange chromatography resolved two major inhibitory peaks, and isoform-specific polyclonal antibodies raised against recombinant PKI alpha and PKI gamma identified these inhibitory activities to be PKI alpha and PKI gamma. A comparison of inhibitory potencies of PKI alpha and PKI gamma expressed in Escherichia coli revealed that PKI gamma was a potent competitive inhibitor of C alpha phosphotransferase activity in vitro (K-i = 0.44 nM) but is 6-fold less potent than PKI alpha (K-i = 0.073 nM). Like PKI alpha, PKI gamma was capable of blocking the nuclear accumulation of Flag-tagged C subunit in transiently transfected mammalian cells. Finally, the murine PKI gamma gene was found to overlap the murine adenosine deaminase gene on mouse chromosome 2. These results demonstrate that PKI gamma is a novel, functional PKI isoform that accounts for the previously observed discrepancy between PKI activity and PKI mRNA levels in several mammalian tissues.