Murine cDNAs representing distinct genes for the regulatory subunits of calmodulin-dependent protein phosphatase (CaM-PrP) were cloned from a testis library, using probes prepared by PCR amplification of brain and testis mRNA. The cDNA sequence of the brain-specific isoform (β1) encodes a 170 amino acid protein (M_r :19.3 kDa), whereas that for the testis isoform (β2) contains 179 residues (M_r 20.7 kDa); these two sequences show 80% amino acid identity. An oligonucleotide probe for the brain isoform hybridized to a single mRNA of 3.6 kilobases (kb) in many tissues, whereas using the β2 probe, two mRNAs of 1.8 and 0.8 kb were detected only in testis. The mRNA for the testis-specific isoform increases markedly during development, its pattern being virtually identical to that of mRNA for a testicular form of the catalytic subunit (α3). These data are consistent with the biological co-regulation of catalytic and regulatory subunits of a testis-specific isoenzyme during germ cell maturation.