A cDNA clone for a type II regulatory (R) subunit of the cAMP-dependent protein kinase (ATP:protein phosphotransferase, EC 18.104.22.168) was isolated from a rat skeletal muscle library using a specific 47-base oligonucleotide probe. The rat cDNA was 1.2 kilobases (kb) in length and contained an open reading frame of 1.113 kb representing 92% of the coding region of the molecule. Nick-translated rat cDNA was then used to isolate a mouse RII cDNA clone from a brain library that contained an open reading frame of 1.143 kb. Because both cDNAs lacked complete coding sequences, the remainder of the RII coding region was obtained from a 15-kb mouse genomic clone. The mouse RII coding region contains 1.2 kb corresponding to a 400-amino acid protein of 51.141 kDa. The mouse cDNA hybridizes to two mRNA species, a 2.4-kb form that was only observed in testis and a 6.0-kb form found in a wide range of tissues, including testis.