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The in vitro effect of therapeutic concentrations of methotrexate on the phagocytosis and intracellular killing of Staphylococcus aureus by circulating human neutrophils was assessed. Neutrophils were isolated from whole blood of six healthy human volunteers by density centrifugation and incubated with 10(-3) M methotrexate. Staphylococcus aureus was opsonized in human serum and added to the prepared neutrophils. Phagocytosis was determined by serial dilutions and plating of unphagocytized bacteria. After treatment with lysostaphin and trypsin, intracellular killing of bacteria was determined at set intervals by serial dilutions and platings of viable bacteria released by neutrophil lysis. Methotrexate-treated neutrophils phagocytized 56% and control neutrophils 67% of bacteria in 15 minutes (P greater than 0.5), and 96% of ingested bacteria were killed in 15 minutes by both populations. In these experiments, the in vitro incubation of circulating human neutrophils with methotrexate produced no significant alterations in neutrophilic phagocytosis or intracellular killing of S. aureus.