Despite its clinical efficacy on intracellular pathogens, the in-vitro intracellular antimicrobial activity of azithromycin, has been shown to be absent or lower than expected from the intracellular concentrations reached. To test the possibility that the high intracellular concentrations of the drug could damage phagocytes, the present study evaluated the effects of azithromycin on (a) the intracellular killing of Staphylococcus aureus by human blood neutrophils (PMNs) and (b) the viability and the respiratory burst of PMNs. Using a fluorochrome assay, we assessed the phagocytosis and intracellular killing of S. aureus by PMNs preloaded with azithromycin, or by PMNs unloaded but with the drug in the culture medium. In addition, possible drug-induced damage to PMNs was evaluated measuring: (a) hydrogen peroxide (H2O2) production and (b) the percentages of PMNs dead at the end of the phagocytosis process. Compared to control PMNs without drug, a time-dependent enhancement in the intracellular killing was observed which was statistically significant after 60 min incubation. The increased intracellular killing was higher in suspensions of unloaded PMNs and azithromycin (P < 0.01) that in suspensions of preloaded PMNs (P < 0.05). This increased intracellular killing was not associated with increased proportions of dead phagocytes, either in preloaded or unloaded PMNs (P < 0.05, each comparison). Similarly no changes in the production of H2O2 by PMNs were observed in the presence of azithromycin. Thus, azithromycin induces a time-dependent increase in the bactericidal activity of human PMNs, without increasing the phagocyte self-killing or modifying H2O2 production.