Phospholipase A2 (PLA2) plays an important pathogenic role in infections caused by several microorganisms and has been implicated in host cell invasion. The mechanism of host cell penetration by the intracellular protozoan Toxoplasma gondii involves several steps; we have investigated the role of PLA2 in cellular invasion by the tachyzoite stage of this parasite. We assayed T. gondii invasion of human fibroblast monolayers by measurement of the selective incorporation of 3H-uracil into growing intracellular parasites. Exogenous PLA2 from snake venom (Naja naja) increased the penetration of fibroblasts by T. gondii, while horse antiserum to Naja hannah venom inhibited penetration. An irreversible PLA2 inhibitor, p-bromophenacyl bromide, blocked penetration without metabolically disabling the parasite. When host fibroblasts were preincubated with this drug, penetration was not affected, supporting a role for parasite rather than host cell PLA2 in the penetration process. Another PLA2 inhibitor, nordihydroguaiaretic acid, also inhibited penetration. We assayed extracellular T. gondii tachyzoites, purified from host cell debris, for PLA2 activity by radiometric detection of fatty acid release from labeled Escherichia coli membranes. Sonically disrupted parasites contained a low level of calciumdependent PLA2 with maximum activity at pH 8.5–9.0. These experiments suggest that a phospholipase is implicated in T. gondii host cell invasion.