Listeria monocytogenes is a bacterial pathogen that multiplies within the cytosol of eukaryotic cells. To identify Listeria genes with preferentially intracellular expression (pic genes), a library of Tn917-lac insertion mutants was screened for transcriptional fusions to lacZ with higher expression inside a macrophage-like cell line than in a rich broth medium. Five pic genes with up to 100-fold induction inside cells were identified. Three of them (purH, purD and pyrE) were involved in nucleotide biosynthesis. One was part of an operon encoding an ABC (ATP-binding cassette) transporter for arginine. The corresponding mutants were not affected in intracellular growth, cell-to-cell spread or virulence, except for the transporter mutant, whose LD50 after intravenous infection of mice was twofold higher than the wild-type. The fifth gene was plcA, a previously identified virulence gene that encodes a phosphatidylinositol-phospholipase C, and is cotranscribed with prfA, a gene encoding a pleiotropic transcriptional activator of known virulence genes. Although plcA expression is known to depend on PrfA, a prfA promoter-lacZ fusion was highly expressed both inside and outside cells. Furthermore, in the presence of cellobiose, a disaccharide recently shown to repress plcA and hly expression, plcA and hly mRNA levels were dramatically reduced without any decrease in the monocistronic prfA mRNA levels. These results demonstrate that virulence gene activation does not depend only on prfA transcript accumulation.