The presence of five distinct isoforms of protein phosphatase 2Cbeta (PP2Cbeta-1 approximately -5) is known. In this study, we demonstrate that the mRNA levels of PP2Cbeta-3, -4 and -5 and PP2Cbeta protein level increased during the course of the first wave of spermatogenesis in neonatal mouse testis. Northern blot and in situ hybridization analyses revealed that PP2Cbeta-3, -4 and -5 were expressed predominantly in pachytene spermatocytes and in more highly differentiated germ cells. The substrate specificity of PP2Cbeta-4 determined with artificial substrates differed from those of PP2Cbeta-3 and -5, suggesting that the difference in the structure of PP2Cbeta-3, -4 and -5 reflect their unique physiological functions in testicular germ cells.