The iap gene of Listeria monocytogenes encodes the extracellular protein p60, which possesses a murein hydrolase activity necessary for septum separation. We constructed L. monocytogenes EGD strains harbouring plasmids that carry the iap gene under the control of the PrfA-regulated promoters of the L. monocytogenes genes hly, mpl, and actA. After insertional inactivation of the chromosomal iap gene in L. monocytogenes EGD, p60 synthesis was strictly dependent on PrfA. Elevated temperature (40 degrees C) enhanced synthesis of p60 in L. monocytogenes when the iap gene was under the control of the hly promoter; this appeared to be associated with increased synthesis of PrfA at this temperature. Synthesis of p60 in L. monocytogenes was significantly lower when the iap gene was placed under the control of the actA or the mpl promoter. Transcription of the iap gene was repressed in L. monocytogenes in the presence of PrfA when iap expression was under the control of the prfA promoter P2. Under the control of the hly promoter the gene produced low levels of secreted p60 in the presence of low amounts of PrfA, and this in turn led to the generation of long listerial cell filaments consisting of bacteria that had failed to separate. Overexpression of p60 in the presence of high levels of PrfA caused formation of single cells, which showed reduced viability depending on the level of secreted p60. These data suggest that the iap gene may be a valuable tool for monitoring virulence gene regulation by PrfA under in vivo conditions, without disturbing the integrity of the infected host cells.