We previously reported that cultured mast cells expressed three discrete phospholipases A2 (PLA2S), one of which showed a remarkable preference for phospholipids bearing an arachidonoyl residue at the sn-2 position [M. Murakami, et al., J. Biochem., 111, 175 (1992)]. In the present study, we have purified and characterized this enzyme using rat mastocytoma RBL-2H3 as an enzyme source. The elution profiles of the arachidonoyl-preferential PLA2 activity of rat mastocytoma RBL-2H3 cells on several column chromatographies were indistinguishable from those of 85-kDa cytosolic PLA2 (cPLA2) characterized so far. The molecular mass of the partially purified PLA2 was estimated to be about 90 kDa by gel filtration and it hydrolyzed arachidonate-containing phospholipids preferentially in the presence of submicromolar Ca2+ concentrations. Furthermore, it was immunoprecipitated with an anti-rabbit cPLA2 antibody almost completely. From these observations, we have concluded that the arachidonoyl-preferential PLA2 in mast cells belongs to the "cPLA2" family.