Using a human cDNA (complementary DNA) encoding the regulatory subunit RI alpha of cAMP-dependent protein kinases (PKA) as a probe, a pseudogene for this PKA isoform was isolated from a human genomic library. The human RI alpha pseudogene was 89% similar to the open reading frame of the expressed human RI alpha at the nucleotide level. Several stop codons were found, indicating that the pseudogene does not encode a functional protein. The pseudogene also contained several frameshift mutations, small deletions, and insertions. No introns were found in the region corresponding to the open reading frame of the expressed RI alpha cDNA. Specific oligonucleotides for the RI alpha gene and pseudogene were constructed and used as primers in polymerase chain reaction (PCR) to amplify human DNA from healthy blood donors. Probing of the PCR products using oligonucleotides specific for the RI alpha gene and RI alpha pseudogene, respectively, showed the presence of both genes in the human genome. When DNA extracted from various somatic cell hybrids was amplified, it was shown that the RI alpha gene was located on human chromosome 17, whereas the RI alpha pseudogene was located in the p21-p31 region on chromosome 1. This is the first report describing a pseudogene for a subunit of cAMP-dependent protein kinases from any species.