The completion of the sequencing of the genomes of both Rickettsia conorii and R. prowazekii provides the opportunity to identify putative virulence factors within these strictly intracellular pathogens. A role for a phospholipase A(2) (PLA(2)) in rickettsial pathogenicity was hypothesized, but the corresponding gene has not been identified. We have identified a gene that encodes a putative phospholipase D (PLD) and that has been detected by Southern blotting in 11 analyzed strains of rickettsiae. The recombinant protein is dimeric and has PLD activity, as demonstrated by its capacity to release [(3)H]-choline from phosphatidyl [(3)H]-choline. This PLD is present in whole rickettsial lysates and likely is a virulence factor, because incubation of rickettsiae with an anti-PLD antibody reduced their cytotoxic activity against Vero cells. This enzyme might account for the activity previously attributed to PLA(2) and might be critical for the intracellular life of these bacteria.