Human cDNA clones for a heat-stable protein kinase inhibitor (PKI) protein of the cAMP-dependent protein kinase (PKA) were isolated using a mouse PKI cDNA fragment. Two human cDNA clones of 1.7 and 2.0 kb were sequenced and shown to encode the entire open reading frame of 228 nucleotides. Together these clones comprised 2147 nucleotides of the mRNA. The deduced amino acid sequence of the human clones showed 100% identity to the rabbit skeletal muscle PKI protein and 97% identity to the mouse brain PKI. The mouse and human PKI cDNAs shared nucleotide homology in their 3' untranslated regions as well as in the 32 nucleotides immediately 5' of the translation initiation site. Northern blot analysis of human skeletal muscle RNA with a human cRNA probe detected a major mRNA of approximately 4.0 kb. Transient overexpression in COS cells verified that a heat-stable inhibitor of protein kinase was produced by he human PKI cDNA, and protein extracts from the transfected COS cells inhibited both the C alpha and C beta isoforms of the PKA catalytic subunit with equal efficacy. Functional expression of the human PKI protein was further studied by assaying the ability of PKI expression vectors to inhibit PKA catalytic subunit stimulation of transcription from the human enkephalin promoter. In these studies, elimination of a conserved alternative translation start site in the 5' untranslated region of PKI was shown to potentiate the inhibitory activity of the PKI expression vector.