In a search for more effective antimicrobial therapy of neonatal Escherichia coli infection, newer beta-lactam antibiotics, cefotaxime and imipenem, were evaluated for their activities against a K1 E. coli strain in vitro and in vivo, and the results were compared with those of conventional therapeutic regimens for neonatal E. coli infection: ampicillin-gentamicin and ampicillin-chloramphenicol. Measured by MICs and MBCs, cefotaxime and imipenem were 8- to 512-fold more active in vitro than the older agents. For in vivo studies, the following daily doses were used: 50 mg/kg for each of imipenem and cilastatin; 100 mg/kg for each of cefotaxime, ampicillin, and chloramphenicol; and 10 mg/kg for gentamicin. At these doses, the mean bactericidal titers in blood and cerebrospinal fluid were significantly greater with newer agents than with ampicillin-gentamicin and ampicillin-chloramphenicol. However, at the doses used, the newer agents were not more effective in vivo than the older agents. This was shown by the similarities in clearance of bacteria from blood and cerebrospinal fluid, incidences of meningitis in bacteremic animals, and mortality rates. Thus, although these two newer antibiotics are more active in vitro and produce greater bactericidal titers in vivo, they do not appear to be superior to conventional regimens for treatment of neonatal E. coli bacteremia and meningitis.