The PrfA transcriptional activator is an essential determinant of Listeria monocytogenes pathogenesis. prfA expression is governed by three differentially regulated promoters: prfAP1 and prfAP2, which are located immediately upstream of prfA coding sequences, and the adjacent plcA promoter via the generation of a plcA-prfA read-through transcript. A series of promoter deletion mutants were constructed to assess the roles of prfAP1 and prfAP2. Elimination of either prfAP1 or prfAP2 resulted in altered regulation of PrfA-regulated genes after in vitro growth. However, these mutants were fully virulent both in an animal model and in tissue culture models of infection, suggesting that the two prfA promoters are functionally redundant in vivo. In contrast, a mutant lacking both prfAP1 and prfAP2 was 100-fold less virulent and was delayed in escape from the host vacuole. Once in the host cytoplasm, however, the double mutant was apparently normal in cell-to-cell spread.