This time-kill study was performed with 65 genetically unique clinical isolates of Gram-negative bacilli and enterococci to further define the antibacterial activity of tigecycline. To our knowledge, this is the largest published time-kill study evaluating tigecycline activity to date. Isolates evaluated were 10 meropenem-resistant Acinetobacter baumannii; 15 Escherichia coli, including 10 extended-spectrum beta-lactamase (ESBL) producers; 15 Klebsiella pneumoniae, including 10 ESBL producers; 20 vancomycin-resistant Enterococcus faecium (VRE), including 10 that were linezolid resistant; and 5 vancomycin-susceptible Enterococcus faecalis. Time-kill testing was performed using tigecycline concentrations of 1x, 2x, and 4x MIC with colony-forming units (CFU) per milliliter determined at 0, 4, 8, 12, 24, 36, and 48 h. Tigecycline MICs (microg/mL) were < or =1 for E. coli and K. pneumoniae, regardless of the isolates' ESBL production; A. baumannii, 0.06 to 4; 9/10 (90%) were < or =2; E. faecalis < or =0.12; and VRE < or =0.25, regardless of linezolid susceptibility. In the time-kill assay, tigecycline significantly inhibited bacterial growth when compared with the growth control. The reduction in growth was <3 log(10) CFU/mL for all isolates, indicative of a bacteriostatic effect.