The susceptibility of 554 recent clinical isolates and known resistant bacterial strains to the new monocyclic beta-lactam Ro 17-2301 were studied and compared to that to other beta-lactams (including aztreonam and temocillin) and gentamicin. Ro 17-2301 had a high degree of activity against the Enterobacteriaceae (MIC90 less than or equal to 0.25 mg/l) being similar or slightly more active than aztreonam and ceftazidime. Strains of Acinetobacter spp. (MIC90 16 mg/l). Haemophilus influenzae strains (including beta-lactamase producers) were more susceptible (MIC90 0.5 mg/l) than those of Neisseria gonorrhoeae (MIC90 4 mg/l); against these latter two groups of isolates aztreonam was more active (MIC90 0.12 mg/l). Both aztreonam and Ro 17-2301 had little activity against Gram-positive cocci with the exception of Streptococcus pneumoniae for which the MIC90 of RO 17-2301 was 16 mg/l. Ro 17-2301 had modest activity against Bacteroides fragilis. The MBC of Ro 17-2301 was very similar to the MIC and the addition of human serum had little effect on the amount of the compound. The mean serum protein binding was 26.3%. A study of the penicillin binding protein affinity of Ro 17-2301 in a strain of Escherichia coli showed PBP 3 to be the primary target. The morphological response to exposure to Ro 17-2301 was filamentation followed by lysis after prolonged exposure.