A full-length complementary DNA (cDNA) clone (pTK-3) encoding an isoform of Mg(2+)-dependent protein phosphatase beta (MPP beta-4) was isolated for the first time from a mouse melanocyte cDNA library. It was strongly suggested that the mRNA corresponding to the pTK-3 insert was a splicing variant of a single pre-mRNA that also encodes MPP beta-1 and -2 (T. Terasawa, T. Kobayashi, T. Murakami, M. Ohnishi, S. Kato, O. Tanaka, H. Kondo, H. Yamamoto, T. Takeuchi, and S. Tamura, 1993, Arch. Biochem. Biophys. 307, 342-349). The amino acid sequence of MPP beta-4 differed from those of MPP beta-1 and -2 only at the carboxyl terminal region. Analysis by reverse transcriptase polymerase chain reaction (RT-PCR) revealed that MPP beta-4 mRNA was expressed only in testis and intestine and not in other mouse tissues tested. Specific expression of the mRNA signals of two other isoforms of MPP beta, MPP beta-3 and -5 (a novel isoform), in testis and intestine was also demonstrated by the RT-PCR. The carboxyl terminal region of MPP beta-5 was found to have a chimera structure composed of part of MPP beta-1 and part of MPP beta-3. The recombinant MPP beta-3 and -4 and the putative MPP beta-5 expressed in Escherichia coli cells exhibited Mg(2+)-dependent and okadaic acid-insensitive protein phosphatase activities. It was demonstrated that the mRNA expression levels of MPP beta-3, -4, and -5 alter according to the maturation of mouse testis. These results suggest that the complex structure of MPP beta isoforms and their tissue- and developmental stage-specific expression reflect the variety of their physiological functions.