From January to May 2000, as part of the Tracking Resistance in the United States Today (TRUST) surveillance initiative, clinical isolates of Enterobacteriaceae (n=2519) and non-fermentative Gram-negatives (n=580) were prospectively collected from 26 hospital laboratories across the United States. Isolates were tested for susceptibility to three fluoroquinolones (ciprofloxacin, levofloxacin, gatifloxacin) and seven other agents. In addition, data for the same period were collected from The Surveillance Network (TSN) Database-USA, an electronic surveillance network that receives data from more than 200 laboratories in the US. Both surveillance methods produced similar results. Against isolates of Enterobacteriaceae, imipenem was the most active agent, followed by the fluoroquinolones; > or = 86.7% of isolates of all species of Enterobacteriaceae except Providencia spp. were susceptible to fluoroquinolones by TRUST and TSN surveillance. TRUST identified differences in susceptibility to the three fluoroquinolones of > or = 2% for Citrobacter spp., Enterobacter cloacae, Proteus mirabilis and Serratia marcescens. Isolates of P. mirabilis were considerably more susceptible to levofloxacin (94.0%) than to ciprofloxacin (87.7%) and gatifloxacin (87.7%). Other results from TRUST included Pseudomonas aeruginosa being slightly more susceptible to ciprofloxacin (73.5%) and levofloxacin (73.0%) than gatifloxacin (71.0%). Imipenem was the only compound with significant activity (95.1% susceptible, TRUST; 87.4% susceptible, TSN) against Acinetobacter baumannii, but it was inactive against Stenotrophomonas maltophilia. S. maltophilia isolates were more susceptible to levofloxacin and gatifloxacin (77.7-79.8%) than ciprofloxacin (29.7-33.0%). Against 513 urinary isolates of Escherichia coli in TRUST, levofloxacin, gatifloxacin and ciprofloxacin were equipotent. Age and gender had no clear effect on the activity of levofloxacin, ciprofloxacin or gatifloxacin. Similar results for all three fluoroquinolones were seen in outpatients and inpatients. TRUST and TSN data indicated that resistance rates had not changed appreciably for any compound studied since a similar study conducted in 1999. TRUST centralized in vitro and electronic (TSN) surveillance methods provided an effective strategy for monitoring trends in resistance.