We studied the inhibitory effects of the fluoroquinolones levofloxacin, ciprofloxacin, and trova-floxacin on growth and extracellular matrix mineralization in MC3T3-E1 osteoblast-like cell cultures. Levo-floxacin had the least inhibitory effect on cell growth, with a 50% inhibitory concentration of approximately 80 μg/ml at 48 and 72 hours. Ciprofloxacin had an intermediate degree of inhibition, with a 50% inhibitory concentration of 40 μg/ml at 48 and 72 hours. Trovafloxacin exerted a profound inhibitory effect on cell growth, with a 50% inhibitory concentration of 0.5 μg/ml, lower than clinically achievable serum levels. The decreased cell counts with up to 2.5 μg/ml of trovafloxacin and with up to 40 μg/ml of ciprofloxacin were not associated with decreased rates of 5-bromo-2′-deoxyuridine incorporation per cell. Alatrovafloxacin, the L-alanyl-l-alanine prodrug of trovafloxacin, exerted effects on proliferation and 5-bromo-2′-deoxyuridine incorporation similar to those of the parent compound. The quinolones evaluated also inhibited extracellular matrix mineralization by MC3T3-E1 cells. Treatment of confluent cultures with trovafloxacin, ciprofloxacin, or levofloxacin resulted in strong inhibition of calcium deposition, as determined on day 14 by alizarin red staining and biochemical analysis. The effect was apparent with 2.5-5 μg/ml of each of the three antibiotics tested and progressively increased to more than a 90% decline in the calcium/protein ratio with 20-40 μg/ml antibiotic concentration. Further in vivo studies are advocated to evaluate the relevance of the in vitro cytotoxicity reported here to bone healing in orthopaedic patients.