Osteomyelitis, osteitis, spondylodiscitis, septic arthritis, and prosthetic joint infections still represent the worst complications of orthopedic surgery and traumatology. Successful treatment requires, besides surgical débridement, long-term systemic and high-concentration local antibiotic therapy, with possible local antibiotic concentrations of 100 microg/ml and more. In this study, we investigated the effect of 20 different antibiotics on primary human osteoblasts (PHO), the osteosarcoma cell line MG63, and the epithelial cell line HeLa. High concentrations of fluoroquinolones, macrolides, clindamycin, chloramphenicol, rifampin, tetracycline, and linezolid during 48 h of incubation inhibited proliferation and metabolic activity, whereas aminoglycosides and inhibitors of bacterial cell wall synthesis did not. Twenty percent inhibitory concentrations for proliferation of PHO were determined as 20 to 40 microg/ml for macrolides, clindamycin, and rifampin, 60 to 80 microg/ml for chloramphenicol, tetracylin, and fluoroquinolones, and 240 microg/ml for linezolid. The proliferation of the cell lines was always less inhibited. We established the measurement of extracellular lactate concentration as an indicator of glycolysis using inhibitors of the respiratory chain (antimycin A, rotenone, and sodium azide) and glycolysis (iodoacetic acid) as reference compounds, whereas inhibition of the respiratory chain increased and inhibition of glycolysis decreased lactate production. The measurement of extracellular lactate concentration revealed that fluoroquinolones, macrolides, clindamycin, rifampin, tetracycline, and especially chloramphenicol and linezolid impaired mitochondrial energetics in high concentrations. This explains partly the observed inhibition of metabolic activity and proliferation in our experiments. Because of differences in the energy metabolism, PHO provided a more sensitive model for orthopedic antibiotic usage than stable cell lines.