We have previously shown that exogenous type II phospholipase A2 (PLA2) alone elicits degranulation of mast cells, including rat serosal mast cells (SMC) and mouse bone marrow-derived mast cells (BMMC). Here we report that lysophosphatidylserine (lysoPS), a co-factor for activation of rodent SMC in response to some tyrosine kinase-coupled agonists, enhanced type II PLA2-elicited histamine release from rat SMC. In contrast, mouse BMMC was insensitive to lysoPS. Our findings demonstrate a novel route for activation of SMC in that type II PLA2 can act as a direct activator of SMC with enhancement by lysoPS, which is generated from membrane phosphatidylserine possibly by the action of the same enzyme.