Ro 09-1428, a new parenteral cephalosporin with a catechol moiety attached at position 7 of the cephalosporin ring, showed high in vitro activity against Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Proteus vulgaris, and Streptococcus pyogenes, with MICs for 90% of strains tested (MIC90s) of less than or equal to 0.39 micrograms/ml. Morganella morganii, Providencia rettgeri, Citrobacter freundii, Haemophilus influenzae, Staphylococcus aureus, and Streptococcus pneumoniae were inhibited with MIC90s of less than or equal to 3.13 micrograms/ml. Serratia marcescens was less susceptible to Ro 09-1428, with a MIC90 of 25 micrograms/ml. The most distinctive feature of Ro 09-1428 was its potent activity against Pseudomonas aeruginosa and Acinetobacter calcoaceticus, with MIC90s of 0.39 and 6.25 micrograms/ml, respectively. Most of the ceftazidime-resistant strains of P. aeruginosa, E. cloacae, and C. freundii were inhibited by Ro 09-1428, while those of S. marcescens were resistant at a concentration of 12.5 micrograms/ml. Ro 09-1428 was more active than ceftazidime against staphylococci. PBP 3 was the most sensitive target in E. coli and P. aeruginosa. The response to ferric iron in growth medium suggests that Ro 09-1428 may be taken up by transport mechanisms similar to those of other catechol cephalosporins. In accordance with its in vitro activity, Ro 09-1428 activity was equal to or greater than ceftazidime activity in efficacy against experimental septicemias in mice. The results indicate that Ro 09-1428 is a broad-spectrum cephalosporin with advantages over ceftazidime in its activity against P. aeruginosa, staphylococci, and ceftazidime-resistant strains of C. freundii and E. cloacae.