To examine whether the in vitro phospholipase activity in Listeria monocytogenes strain H4 was due to two nucleotide mutations (C to T at position -26 and A to G at position +1) in plcB or resulted from regulatory activation, two mutants H4-plcB-m1 (single mutation at position -26) and H4-plcB-m2 (substitution at both positions) were constructed by site-directed mutagenesis. It was found that the two mutants had significantly lower transcription of plcB than their parent strain H4 and did not show phospholipase activity on the egg yolk agar, implying that the apparent phospholipase activity of strain H4 could be related to single substitution at position -26 of plcB, most probably by its 5'-untranslated region (5'-UTR) regulation mechanism. Tn917-based transposon mutagenesis generated eight L. monocytogenes mutants lacking phospholipase activity among 560 mutant candidates. Seven mutants had transposon insertion into prfA (encoding positive regulatory factor A) open reading frame, whereas only one mutant (WF-L127) was inserted into the P1 promoter region of prfA (prfAP1). Transcription of major virulence genes was significantly lower in both types of mutants than in their parent strain H4. Disruption of prfAP1 in WF-L127 abolished its phospholipase C activity but did not change its hemolytic phenotype, indicating that plcB was more dependent on prfA regulation than hly. Taken together, this study presents some evidence for the regulation of plcB expression by its 5'-UTR mechanism.