A competitive reverse transcription-PCR method was developed for the semiquantitation of the expression of genes encoding bicomponent leucotoxins of Staphylococcus aureus, e.g., Panton-Valentine leucocidin (lukPV), gamma-hemolysin (hlgA and hlgCB), and LukE-LukD (lukED). The optimization procedure included RNA preparation; reverse transcription; the use of various amounts of enzymes, antisense primer, and RNA; and the final amplification chain reaction. Reproducible results were obtained, with sensitivity for detection of cDNA within the range of 1 mRNA/10 4 CFU to 10 2 mRNA/CFU, depending on the gene. Both specific mRNAs were more significantly expressed at the late-exponential phase of growth. Expression was about 100-fold higher in yeast extract-Casamino Acids-pyruvate medium than in heart infusion medium. Expression of the widely distributed gamma-hemolysin locus in the NTCC 8178 strain was around 10-fold diminished compared with that in the ATCC 49775 strain. Because of the lower level of hlgA expression, the corresponding protein, which is generally not abundant in culture supernatant, should be investigated for its contribution to the leucotoxinassociated virulence. The agr, sar, and agr sar mutant strains revealed a great dependence with regard to leucotoxin expression on the global regulatory system in S. aureus, except that expression of hlgA was not affected in the agr mutant.Staphylococcal bicomponent leucotoxins are exotoxins consisting of two nonassociated but synergic class S (31 to 32 kDa) and class F (35 kDa) proteins. Among this family of toxins, the Panton-Valentine leucocidin (PVL) is encoded by two contiguous and cotranscribed genes, lukFPV and lukS-PV (31). Another locus encodes ␥-hemolysin and comprises three genes: the first two encode class S proteins (HlgA and HlgC), and the third one encodes a class F protein (HlgB). hlgA constitutes an upstream open reading frame, whereas hlgC and hlgB (hlgCB in this text) are cotranscribed (10). Another locus was recently characterized as two cotranscribed class S and class F proteinencoding genes, lukE and lukD (lukED in the text), respectively (16). Production of leucotoxins among Staphylococcus aureus strains was studied by radial gel immunoprecipitation (16, 30), but quantitation of leucotoxin expression by enzyme-linked immunosorbent assay remains difficult because of cross-reactivity due to sequence identity between class S components (55 to 72%) and class F components (71 to 79%), respectively (29). There may be S. aureus strains that produce ␥-hemolysin, PVL, and/or LukE-LukD.Leucotoxin production was associated with infections resulting in furuncles, community pneumonia, and some antibioticassociated diarrhea (11,15,24). These infection-related leucotoxins act as activators of human neutrophils before creating lytic pores sensitive to monovalent cations (34). The leucotoxins were shown to induce an important inflammatory response in vivo in rabbit skin and in rabbit vitreous humor (16,31,32). Therefore, to determine their respective roles in...