The chemotactic response of human neutrophils was examined in the presence of tetracycline. High concentrations of the antibiotic decreased chemotaxis in vitro but did not have an inhibitory effect on the ability of neutrophils to respond to the chemotactic components of human complement. Mixtures of human serum and endotoxin had decreased chemotactic ability when they were preincubated with various concentrations of tetracycline, the decrease being concentration dependent. This inhibitory effect of tetracycline on the chemotactic response of human neutrophils was not time dependent. Eight different tetracycline compounds were found to produce similar inhibitory effects on the chemotactic response of human neutrophils in vitro. The results indicated that tetracycline either inhibits the generation of neutrophil chemotactic factors of human serum or binds with the chemotactic factors after their generation to cause inactivation.