Microbial pathogens use a number of genetic strategies to invade the host and cause infection. These common themes are found throughout microbial virulence factors. Secretion of enzymes, such as phospholipase, has been proposed as one of these themes which is used by bacteria, parasite, and pathogenic fungi. The role of extracellular phospholipase as a potential virulence factor in pathogenic fungi, including Candida albicans, Cryptococcus neoformans and Aspergillus has gained credence recently. In this address data implicating phospholipase as a virulence factor in Cryptococcus neoformans and Aspergillus fumigatus will be presented. This will be followed by a more detailed description of our molecular and biochemical approaches we used to more definitively delineate the role of phospholipase in the virulence of C. albicans. First, we purified the phospholipase B protein, the dominant phospholipase secreted by C. albicans, obtained the amino acid sequence of its N-terminus and an internal peptide fragment, and used this information to clone the gene encoding the protein using a PCR-based approach. Nucleotide sequence analysis revealed an ORF of 1818 bp that predicted for a pre-protein of 605 amino acid residues. The deduced amino acid sequences of the cloned gene (PLB 1) showed 42.3%, 45%, and 47.8% overall sequence identity, with the reported sequences of phospholipase B cloned from Penicillium notatum, Saccharomyces cerevisiae, and Saccharomyces rosei, respectively. Second, using targeted gene disruption, URA blaster, we created C. albicans null mutants which failed to secrete phospholipase B. Third, we tested the ability of these isogenic strain pairs to cause lethality using a murine model of hematogenously disseminated candidiasis. Our data demonstrate that the parent phospholipase-producing strain caused more fatality in mice, while the null phospholipase-deficient strain was avirulent. Importantly, the parent and null mutants had similar growth and germination rates. These data prove that phospholipase B is essential for candidal virulence, and pave the way for studies directed at determining the mechanism/s through which phospholipase modulate candidal virulence. Understanding phospholipase as a common theme in fungal pathogenicity is critical for developing new antifungal strategies based on anti-virulence.