ABSTRACT The ability of Legionella pneumophila to cause Legionnaires’ disease is dependent on its capacity to survive in the intracellular environment of its host cells. Furthermore, outbreaks of this disease have been associated with contaminated water sources where L. pneumophila survives as a parasite of protozoa. In this study, we determined the effect of nutritional auxotrophy on the ability of L. pneumophila to survive in the intracellular environment of its host cells. We generated a diaminopimelic acid (DAP) auxotroph (AA400) of L. pneumophila by disruption of the aspartate-β-semialdehyde ( asd ) gene. The ability of AA400 to survive within macrophages and protozoa was found to be defective. This defect was due solely to the asd disruption since complementation of the mutant with the wild-type asd gene restored its capacity for intracellular survival. Furthermore, the defect was not completely complemented by DAP supplementation to the culture media. Thus, our results suggest that disruption of the asd gene may prove to be useful in the design of attenuated vaccines against Legionnaires’ disease.