We observed that glutamate greatly enhances the survival of Listeria monocytogenes in gastric fluid, a phenomenon that is directly linked to glutamate decarboxylase activity (GAD). Glutamate-mediated acid tolerance has been associated in other intestinal genera with the GAD system, in which glutamate is internalized and converted to gamma-aminobutyrate (consuming an intracellular proton) that is subsequently exchanged for another extracellular glutamate via a membrane-located antiporter. Molecular analysis of L. monocytogenes LO28 revealed the presence of two glutamate decarboxylase homologues, designated gadA and gadB, that are differentially expressed. The gadB gene is co-transcribed in tandem with an upstream gene, gadC, which encodes a potential glutamate/gamma-aminobutyrate antiporter. Expression of this transcript is upregulated in response to mild acid stress (pH 5.5). In contrast, expression of the monocistronic gadA message was weaker and was not induced by mild acid treatment. Non-polar deletion mutations resulted in a dramatic decrease in the level of GAD activity and a concomitant decrease in acid resistance in the order LO28 > DeltagadA > DeltagadB = DeltagadC > DeltagadAB for both stationary and logarithmic phase cultures. The exquisite sensitivity of the DeltagadAB mutant to ex vivo porcine and synthetic human gastric fluid demonstrates a clear role for the GAD system in facilitating survival of the organism in the stomach after ingestion and in other low-pH environments. Furthermore, variations in levels of GAD activity between different strains of L. monocytogenes correlate significantly with levels of tolerance to gastric fluid. Sensitive strains, which include the sequenced L. monocytogenes EGD, exhibit reduced levels of GAD activity. It is clear from this study that expression of GAD by L. monocytogenes strains is an absolute requirement for survival in the stomach environment.