The Listeria monocytogenes genome contains more than 20 genes that encode cell surface-associated internalins. To determine the contributions of the alternative sigma factor sigma(B) and the virulence gene regulator PrfA to internalin gene expression, a subgenomic microarray was designed to contain two probes for each of 24 internalin-like genes identified in the L. monocytogenes 10403S genome. Competitive microarray hybridization was performed on RNA extracted from (i) the 10403S parent strain and an isogenic Delta sigB strain; (ii) 10403S and an isogenic Delta prfA strain; (iii) a (G155S) 10403S derivative that expresses the constitutively active PrfA (PrfA*) and the Delta prfA strain; and (iv) 10403S and an isogenic Delta sigB Delta prfA strain. Sigma(B)- and PrfA-dependent transcription of selected genes was further confirmed by quantitative reverse-transcriptase polymerase chain reaction. For the 24 internalin-like genes examined, (i) both sigma(B) and PrfA contributed to transcription of inlA and inlB, (ii) only sigma(B) contributed to transcription of inlC2, inlD, lmo0331, and lmo0610; (iii) only PrfA contributed to transcription of inlC and lmo2445; and (iv) neither sigma(B) nor PrfA contributed to transcription of the remaining 16 internalin-like genes under the conditions tested.