We have isolated a cDNA clone corresponding to the Mr 38,000 catalytic subunit of bovine type 2A protein phosphatase. The cDNA was isolated from a bovine adrenal gland cDNA library through the use of oligonucleotide probes whose sequences were based on partial amino acid sequence obtained from cyanogen bromide fragments of the purified cardiac enzyme. The entire 1724-base-pair cDNA has been sequenced and found to contain an open reading frame coding for a protein of 325 amino acids having a calculated molecular weight of 37,320. The deduced amino acid sequence contains the experimentally determined sequences of five different cyanogen bromide peptides. Transfection of COS-m6 cells with the cloned cDNA resulted in transient expression of a protein that could be detected by immunoblot analysis with a monoclonal antibody directed against the purified cardiac protein phosphatase. The expressed protein had the same apparent molecular weight as the purified enzyme when analyzed by NaDodSO4/polyacrylamide gel electrophoresis, suggesting that this clone contains the entire coding region of the phosphatase mRNA. The cloned cDNA hybridizes to a mRNA of 2.0 kilobases in bovine heart and adrenal gland. Under conditions of reduced stringency, the cDNA also hybridizes to a mRNA species of 1.2 kilobases in cardiac tissue.