The in vitro activity of levofloxacin was investigated against 256 clinical strains of four gram-positive genera (Staphylococcus, Streptococcus, Enterococcus, and Listeria). Ofloxacin and ciprofloxacin were used as comparators. Uniform susceptibility to levofloxacin was recorded among methicillin-susceptible staphylococci, streptococci other than Streptococcus agalactiae, regardless of their being susceptible, intermediate, or resistant to penicillin (S. pneumoniae) or erythromycin (S. pyogenes and S. pneumoniae), in enterococci other than Enterococcus faecalis and E. faecium, and in Listeria monocytogenes isolates. Moreover, 1 of 22 S. agalactiae isolates and 1 of 19 E. faecium isolates was resistant, and 2 of 19 were intermediate. Resistances to levofloxacin with MIC90s in the resistance range were only observed in methicillin-resistant staphylococci and E. faecalis isolates. In any case, the MICs of ofloxacin and ciprofloxacin were usually 2-4 times higher than those of levofloxacin. In time-kill assays using three test strains (a methicillin-susceptible Staphylococcus aureus isolate, a penicillin-susceptible Streptococcus pneumoniae isolate, and an E. faecalis isolate), the bactericidal activity of levofloxacin was greater than that of ciprofloxacin.