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A unique isoform of the catalytic subunit of calmodulin-dependent protein phosphatase (CaM-PrP) was cloned from a murine testis library. The cDNA sequence of 1964 base pairs contained an open reading frame encoding a protein of 513 amino acids (Mr approximately 58,706), the predicted isoelectric point of which (pI 7.1) was much more basic than those of brain isoforms (pI 5.6-5.8). The deduced amino acid sequence was 77-81% identical to two other murine CaM-PrP genes and displayed a distinct Southern blot hybridization pattern, indicating that it was derived from a separate gene (type 3). High amounts of a 2800-nucleotide mRNA transcript were observed in testis, whereas mRNA species were not detectable in brain; thus, it seems likely that this CaM-PrP represents a nonneural isoenzyme. Measurements of CaM-PrP mRNA during testicular development showed a dramatic increase in expression during weeks 4-6, correlating with the later stages of spermatogenesis. These data suggest that this phosphatase isoform may be involved in germ-cell function and are consistent with the report of a flagellum-associated form of CaM-PrP that may regulate sperm motility [Tash, J. S., Krinks, M., Patel, J., Means, R. L., Klee, C. B. &amp; Means, A. R. (1988) J. Cell Biol. 106, 1625-1633].