Pneumolysin has been identified as a virulence factor in Streptococcus pneumoniae disease. In addition to producing tissue injury through its cytolytic effect, pneumolysin might injure tissues indirectly by eliciting an inflammatory response. We demonstrate for the first time that pneumolysin is a rapid and potent activator of cellular phospholipase A in bovine pulmonary artery endothelial cells. In contrast to other toxin-activated phospholipases, pneumolysin-stimulated phospholipase A showed no substrate specificity among major cellular membrane phospholipids. Phospholipase A activation required the formation of functional transmembrane pores by pneumolysin rather than membrane lipid perturbation. Pneumolysin stimulation of phospholipase A was calcium dependent; however, pneumolysin did not appear to function simply as a calcium ionophore. Pneumolysin was capable of stimulating purified bee and snake venom phospholipase A2s against a phospholipid substrate isolated from endothelial cells. Thus, pneumolysin stimulates cellular phospholipase A and the resulting products might further injure tissues by direct cytolytic effect or by evoking inflammatory responses.