In the mammalian brain, there are multiple catalytic subunits for the Ca(2+)- and calmodulin-dependent protein phosphatase [also called protein phosphatase 2B (PP-2B) and calcineurin] that are derived from two structural genes. The coding sequences of these two genes are distinguished by the absence (PP2B alpha 1) or the presence (PP2B alpha 2) of an amino terminus containing polyproline. Both of these genes can produce intragenic isoforms through alternative splicing. In the present study, a potential phylogenetic relationship of these genes was inferred from analysis of genomic DNA and from studies of mRNA and protein expression. Southern blot analysis showed unique restriction fragments for both genes in seven mammalian species; however, in organisms from two nonmammalian vertebrates (chicken and lizard), hybridization was observed only for PP2B alpha 1. In agreement with these results, Northern blots of mammalian brain RNA showed transcripts for both genes, with about two to three times more of the PP2B alpha 1 mRNAs, whereas in chicken and lizard, only PP2B alpha 1 transcripts were detected. An analysis of protein expression by two-dimensional electrophoresis was also consistent with these findings. For the purified mammalian brain protein, eight to ten variants were observed with isoelectric points of 5.2-5.8; immunoblot analysis using anti-peptide antibodies indicated that the majority of these were PP2B alpha 1 forms. In chicken brain, multiple isoforms were recognized by antibodies against the PP2B alpha 1 forms, but no reactivity was seen with those against the PP2B alpha 2 forms. Taken together, these findings suggest that: (i) in mammals, the predominant catalytic subunit isoforms in brain are PP2B alpha 1 products and (ii) the gene for the polyproline-containing catalytic subunit of calmodulin-dependent phosphatase (PP2B alpha 2) may have evolved after the avian/reptilian branching point, perhaps to carry out a role(s) of particular significance in mammals.