The stringent response is a global bacterial response to stress that is mediated by accumulation of the alarmone (p)ppGpp. In this study, treatment with mupirocin was shown to induce high levels of (p)ppGpp production in Enterococcus faecalis, indicating that this nosocomial pathogen can mount a classic stringent response. In addition, (p)ppGpp was found to accumulate in cells subjected to heat shock, alkaline shock, and inhibitory concentrations of vancomycin. Sequence analysis of the E. faecalis genome indicated that (p)ppGpp synthesis is catalyzed by the bifunctional synthetase/hydrolase RelA and the RelQ small synthase. The (p)ppGpp profiles of DeltarelA, DeltarelQ, and DeltarelAQ strains revealed that RelA is the major enzyme responsible for the accumulation of (p)ppGpp during antibiotic or physical stresses, while RelQ appears to be responsible for maintaining basal levels of alarmone during homeostatic growth. Compared to its parent, the DeltarelA strain was more susceptible to several stress conditions, whereas complete elimination of (p)ppGpp in a DeltarelAQ double mutant restored many of the stress-sensitive phenotypes of DeltarelA. Interestingly, growth curves and time-kill studies indicated that tolerance to vancomycin is enhanced in the DeltarelA strain but diminished in the DeltarelQ and DeltarelAQ strains. Finally, virulence of the DeltarelAQ strain but not of the DeltarelA or DeltarelQ strain was significantly attenuated in the Caenorhabditis elegans model. Taken together, these results indicate that (p)ppGpp pools modulate environmental stress responses, vancomycin tolerance, and virulence in this important nosocomial pathogen.