Pyruvate dehydrogenase phosphatase (POP) is a dedicated mitochondrial enzyme responsible for the regulation of the pyruvate dehydrogenase complex (PDC). Here we report the molecular cloning of two cDNAs encoding isozymes of rat PDP. The first cDNA is highly similar to the previously identified bovine PDP (PDP1). The second cDNA encodes for a protein (PDP2) that is highly homologous to its bovine and rat counterparts. Analysis of the deduced protein sequence of PDP2 revealed approximately 70% similarity with both rat and bovine PDP1. The sequence of the first 60-70 amino acids of PDP2 is consistent with mitochondrial uptake sequences, suggesting its mitochondrial localization. Like other metal-dependent Ser/Thr phosphatases. the new enzyme has all the residues involved in the formation of the active site, but lacks the helix-loop-helix Ca2+-binding motif responsible for the regulation of PDP1 activity by Ca2+. Both isozymes, obtained as recombinant proteins, preferen tially used phospho-PDC as a substrate and did not show substantial activity toward the related branched chain a-ketoacid dehydrogenase. The specific activity of PDP2 was comparable with Ca2+-stimulated activity of rat PDPl.but it had substantially higher KM for Mg2+ and did not show any activation by Ca2+. Thus, for the first time these results establish that in mammals PDP is a multi gene family and imply that the different isozymes may have specialized functions in the regulation of PDP activity. Supported by PHS grants GM r>1262 and DK 47844.