Production of phosphatidylinositol-specific phospholipase C (PIPLC), an exoenzyme of some strains of Staphylococcus aureus, has been epidemiologically associated with virulence. To investigate the elaboration of S. aureus PIPLC, we have evaluated in vitro conditions that maximize production of enzymatically active PIPLC. PIPLC activity was assessed by measuring the release of H-3-inositol-phosphate from the substrate H-3-phosphatidylinositol. Lowering the pH of S. aureus cultures from 7.0 to 5.4 progressively increased the yield of PIPLC. The final yield of PIPLC was at least five-fold greater when the initial culture pH was 5.4 compared with 7.0. Low pH enhanced PIPLC activity recovered from two S. aureus strains capable of high PIPLC production, but not from a strain producing little PIPLC. At both pH 5.0 and 7.4, PIPLC production peaked during mid- to late-logarithmic phase. We conclude that an acidic starting pH of culture media increases the yield of PIPLC activity elaborated during active growth of S. aureus.