The in vitro activities of the new ureidopenicillins piperacillin, mezlocillin, azlocillin, and Bay k 4999 were compared with those of ampicillin and ticarcillin against 336 Enterobacteriaceae, 109 nonfermenters, 55 Neisseria, and 28 Haemophilus influenzae isolates. Bay k 4999 displayed the largest spectrum of activity and had lower minimal inhibitory concentrations than any of the other penicillins against all of the species tested. Piperacillin showed the same spectrum but was slightly less active than Bay k 4999; it was slightly more effective than mezlocillin against Enterobacteriaceae and fully as active as azlocillin against Pseudomonas. All ureidopenicillins were substantially more active than ampicillin and ticarcillin. Isolates highly resistant to ampicillin or ticarcillin were also less susceptible to the ureidopenicillins.