Tetracycline, in concentrations common during therapy, markedly depressed migration of human leukocytes in vitro. Sera from 12 of 13 volunteers who received oral tetracycline after infection with Mycoplasma pneumoniae inhibited leukotaxis of normal leukocytes. Random migration and chemotaxis of leukocytes from two additional subjects were depressed for up to 24 hr after a single 1-g dose of tetracycline. When tetracycline was tested over a wide range of concentrations, leukotaxis was depressed by lower concentrations (0.01–10 µg/ml) but was stimulated by higher concentrations (30–300 µg/ ml) of the antibiotic. Metabolic studies revealed that production of leukocyte lactate was elevated significantly in the presence of a high level of tetracycline. The mechanisms by which tetracycline affects leukotaxis are not known.