Optimal therapy of infections caused by bacteria able to survive within phagocytes requires the use of antibiotics which inactivate these intracellular organisms. To define characteristics that determine entry of antimicrobial agents into phagocytes, we studied the uptake of 14 radiolabeled antibiotics by rabbit AM. Cell-antibiotic mixtures were incubated for 2 hr, and at intervals antibiotic uptake was determined by velocity-gradient centrifugation (separation of cells from extracellular antibiotic). Many drugs failed to penetrate AM readily. Cellular concentrations of penicillin G and three cephalosporin antibiotics were much lower than extracellular levels (C/E = less than 0.1 to 0.4). Gentamicin, isoniazid, and tetracycline attained C/E values of 0.5 to 0.8. The more lipid-soluble antibiotics, refampin, lincomycin, and chloramphenicol, were concentrated approximately twofold (C/E = 2) in AM. Ethambutol (C/E = 7) and two erythromycin preparations (C/E = greater than 20) were markedly accumulated by macrophages. In comparison with other antibiotics tested, the uptake of clindamycin was both massive and rapid (C/E = 50 by 30 min). Ethambutol, erythromycin and clindamycin uptakes by AM are dependent upon oxidative metabolic processes. Detailed characterization of clindamycin uptake confirmed that the drug is accumulated by an active transport system. These findings, in association with studies of antibiotic-mediated influence on phagocytes, should provide information useful in establishing guidelines for optimal antibiotic usage.