The in vitro activity of GR69153, a new catechol-substituted cephalosporin, was compared with those of ceftazidime, imipenem, meropenem, and ceftriaxone against 604 recent clinical isolates and other strains with known mechanisms of resistance. The MICs of GR69153 for 90% of the members of the family Enterobacteriaceae tested were less than 0.5 micrograms/ml, with the exceptions of those for Serratia spp. (4 micrograms/ml), Citrobacter spp. (2 micrograms/ml), and Enterobacter spp. (8 micrograms/ml). Ninety percent of Pseudomonas aeruginosa isolates were susceptible to less than or equal to 1 microgram of GR69153 per ml. With the exception of methicillin-resistant strains, 90% of Staphylococcus aureus isolates were susceptible to less than or equal to 2 micrograms/ml, and GR69153 was four- to eightfold more active than ceftazidime and ceftriaxone against these strains. Isolates of Haemophilus influenzae, Branhamella catarrhalis, Neisseria spp., and Streptococcus pneumoniae (penicillin susceptible) were highly susceptible (MIC for 90% of the strains, less than or equal to 0.12 micrograms/ml). GR69153 was stable to hydrolysis by the TEM-1 and TEM-5, SHV-1 and SHV-2, and K1 beta-lactamases, but some susceptibility to hydrolysis by the TEM-3, TEM-9, and P99 enzymes was observed. The protein-binding activity of GR69153 was 74.5 to 66.8%, depending on the concentration, and serum had little effect upon activity.