Burkholderia pseudomallei is a serious bacterial pathogen that can cause a lethal infection in humans known as melioidosis. In this study two of its phospholipase C (PLC) enzymes (Plc-1 and Plc-2) were characterized. Starting with a virulent strain, two single mutants were constructed, each with one plc gene inactivated, and one double mutant with both plc genes inactivated. The single plc mutants exhibited decreased extracellular PLC activity in comparison to the wild-type strain, thereby demonstrating that the two genes encoded functional extracellular PLCs. Growth comparisons between the wild-type and PLC mutants in egg-yolk-supplemented medium indicated that both PLCs contributed to egg-yolk phospholipid utilization. Both PLCs hydrolysed phosphatidylcholine and sphingomyelin but neither was haemolytic for human erythrocytes. Experimental infections of eukaryotic cells demonstrated that Plc-1 itself had no effect on plaque-forming efficiency but it had an additive effect on increasing the efficiency of Plc-2 to form plaques. Only Plc-2 had a significant role in host cell cytotoxicity. In contrast, neither Plc-1 nor Plc-2 appeared to play any role in multinucleated giant cell (MNGC) formation or induction of apoptotic death in the cells studied. These data suggested that PLCs contribute, at least in part, to B. pseudomallei virulence and support the view that Plc-1 and Plc-2 are not redundant virulence factors.