The ability of human neutrophils to aid in defense against pulmonary infection with Mycobacterium tuberculosis is controversial. In this study, we have shown that neutrophils respond to and phagocytose M. tuberculosis in human lesions. Neutrophils from healthy individuals were able to kill significant fractions of an inoculum of M. tuberculosis within 1 h of phagocytosis, and this ability was enhanced by tumor necrosis factor alpha but not by gamma interferon. The mycobactericidal mechanism was nonoxidative, as inhibitors of reactive oxygen or reactive nitrogen intermediates did not interfere with killing. However, the mycobactericidal mechanism was associated with increased exposure of intracellular M. tuberculosis to neutrophil defensins. In vitro, human neutrophil peptides 1 to 3 were not able to kill the bacilli even at much higher levels. These studies support the concept that human neutrophils are directly involved in defense against infection with M. tuberculosis.