A Ca2+-dependent lysophospholipase D activity in microsomal preparations from the rabbit kidney medulla hydrolyzes the choline moiety from 1-O-[9,10-3H]hexadecyl-2-lyso-sn-glycero-3-phosphocholine (lyso-PAF) to form 1-O-[9,10-3H]hexadecyl-2-lyso-sn-glycero-3-P; the latter is subsequently dephosphorylated by a phosphohydrolase to 1-O-[9,10-3H]hexadecyl-sn-glycerol. Sodium vanadate, which is known to inhibit phosphohydrolases, reduces the proportion of hexadecylglycerol and increases the formation of hexadecyl-lysoglycerophosphate. Essentially no hydrolysis occurs when the sn-2 position of the hexadecyllysoGPC substrate contains an acyl moiety. The lysophospholipase D in rabbit kidney is of microsomal origin and has a broad pH optimum between 8.0 and 8.8, with the activity decreasing sharply from pH 7.6 to 7.2. Wykle et al. (Biochim. Biophys. Acta 619 (1980) 58-67) have previously demonstrated the existence of a microsomal lysophospholipase D (specific for ether lipid substrates) in rat tissues that requires Mg2+ and exhibits a pH optimum of 7.2; high activities of the Mg2+-dependent lysophospholipase D were found in liver and brain, but not in kidney. In contrast to the Mg2+-dependent lysophospholipase D in rat tissues, the renal enzyme from rabbits requires Ca2+ (5 mM), whereas Mg2+ (5 mM) exhibits little stimulatory action. Under optimal assay conditions (0.1 M Tris-HCl (pH 8.4)/5 mM CaCl2), lysophospholipase D in the rabbit kidney medulla has an activity of 2.7 nmol/min per mg protein compared to 0.9 nmol/min per mg protein for the lysophospholipase D in the rat kidney medulla (0.1 M Tris-HCl (pH 7.2)/5 mM MgCl2). The Ca2+-dependent lysophospholipase D is highest in the liver and kidney medulla from rabbits, but is very low in rat tissues; similar activities were found in male and female rabbits. Our data indicate that the divalent metal ion requirements for expression of maximum lysophospholipase D activities can differ markedly among animal species and also suggest the microsomal Ca2+-dependent lysophospholipase D is an important catabolic route for lyso-PAF metabolism in rabbit renomedullary tissue.