To learn more about the effects of smokeless tobacco on the defensive functions of neutrophils, we studied the influence of nicotine on these cells in vitro, looking at their bactericidal activity against oral pathogens, and at their ability to produce microbicidal reactive oxygen species (oxygen radicals). Exposure of human blood neutrophils to nicotine (0.01% to 0.1%) inhibited their ability to kill Actinomyces naeslundii, Actinobacillus actinomycetemcomitans, and Fusobacterium nucleatum. Although these concentrations of nicotine are high, such concentrations are relevant to phagocytes in the gingival sulcus, because smokeless tobacco contains 0.5% to 3.5% nicotine by dry weight. Nicotine had no such inhibitory effect when the killing assay was performed in an anaerobic environment, implying that nicotine preferentially affected oxygen-dependent killing mechanisms. To further investigate the effects of nicotine on production of oxygen radicals, neutrophils were primed with lipopolysaccharide and triggered with f-met-leu-phe or phorbol ester in the presence of nicotine. Nicotine inhibited production of superoxide anion (measured by reduction of cytochrome c) and hydrogen peroxide (measured by oxidation of phenol red). Nicotine inhibition of superoxide production was reversible by washing away the nicotine. By observing that nicotine inhibited the reduction of cytochrome c by reagent potassium superoxide, we determined that nicotine directly absorbed superoxide. In addition, by examining nicotine inhibition of the uptake of oxygen by neutrophils, we determined that nicotine also interfered with the production of oxygen radicals by these cells. Nicotine also inhibited production of superoxide and interleukin-1 beta by monocytes. Nicotine did not affect the viability of neutrophils and monocytes, as determined by their ability to exclude trypan blue dye. Inhibition of the aerobic antimicrobial functions of neutrophils and monocytes by nicotine may alter the microbial ecology of the oral cavity, and this might be one mechanism by which nicotine compromises the oral health of users of tobacco products.