Entry of Listeria monocytogenes into epithelial cells requires expression of inIA, the first gene of an operon comprising two genes: inIA, which encodes internalin, a 800-amino-acid protein, and inIB, which encodes a 630-amino-acid protein. We report here that the inI locus is transcribed on two transcripts in constant relative ratio: a 5 kb transcript spanning inIA and inIB, and a 2.9 kb transcript that covers only inIA. The promoter is located 397 bp from the GTG initiator of inIA and displays in its -35 region a palindrome similar to that found in promoters controlled by the pleiotropic activator prfA. Transcription of the inI locus is, as are several other L. monocytogenes virulence genes, activated by prfA and regulated by temperature--with higher expression at 37 degrees C versus 25 degrees C--and bacterial growth state. It is maximal during exponential growth and correlates with maximal invasivity of the bacteria in the human epithelial cell line Caco-2. It also correlates with maximum amounts of internalin present on the bacterial surface. Internalin is also detected in substantial amounts in culture supernatants. Taken together, these data suggest that surface-bound internalin plays an important role in bacterial entry but do not exclude a role for the released form.