Two human isoforms of membrane associated phosphatidic acid phosphatase have been described (PAP-2a and -2b), and both enzymes have been shown to have broad substrate specificity and wide tissue distribution [Kai et al., J. Biol. Chem. 272 (1997) 24572-24578]. With this report we describe a third isoform, PAP-2c, that we found by searching the database of expressed sequence tags (dbEST) with PAP-2a and PAP-2b sequences. Key structural features described previously in PAP-2a and -2b, including the glycosylation site, putative transmembrane domains, and the proposed catalytic site, are conserved in the novel phosphatase. The kinetics of the three enzymes were compared using as substrates phosphatidic acid, lysophosphatidic acid, and N-oleoyl ethanolamine phosphatidic acid. Km values for each of the substrates, respectively, were (in microM) PAP-2a: 98, 170, 116; PAP-2b: 100, 110, 56; and PAP-2c: 150, 340, 138. Expression of PAP-2c mRNA is more restricted than the two previously described isoforms.