The interaction between human erythrocyte lysates and antibiotics was studied, and the effect of intracellular components on the activity and binding of the drugs was determined. Lysates inhibited antibacterial activity of penicillin G, dicloxacillin, tetracycline, and minocycline to about the same extent as did human plasma. Dicloxacillin activity was the most inhibited, followed by the activities of penicillin G, minocycline, and tetracycline. All four antibiotics bound to human hemoglobin, as determined by gel filtration methods. Heme-free globin was also effective in binding the antibiotics. In addition, minocycline and tetracycline were bound to another erythrocytic protein, which, on the basis of electrophoretic mobility, molecular size, and localization, has been identified as carbonic anhydrase. Experiments with pure preparations of carbonic anhydrase revealed that the C isozyme is the major binder of the tetracyclines and that zinc is required for binding. Tetracyclines did not inhibit enzymatic activity of carbonic anhydrase.