The in vitro activities of mecillinam, a new beta-amidinopenicillin, and ampicillin, alone and in combination, against an Escherichia coli strain and a Klebsiella pneumoniae strain were compared, and these results were correlated with their respective activities in vivo in experimental meningitis. The mecillinam-ampicillin combination was synergistic in vitro against both strains when tested by a modified checkerboard technique (bacteriostatic synergy). However when quantitative bactericidal synergy studies were made, the relative bactericidal rate of the combination was more rapid than that of either drug alone ("bactericidal synergy") against the Escherichia coli isolate only. In a rabbit model of Enterobacteriaceae meningitis, in vivo bactericidal activity correlated with results obtained in vitro. Both drugs were administered by continuous intravenous infusion for 8 h. Serum and cerebrospinal fluid antibiotic levels were similar to those achieved in humans. Cerebrospinal fluid bacterial concentrations (colony-forming units [CFU] per milliliter) were quantitatively titrated at 2-h intervals. Both drugs, alone or the combination, were ineffective against the K. pneumoniae strain in vivo (change in titer <1 log in 8 h). In contrast, the combination produced a markedly enhanced bactericidal effect against the E. coli strain (mean +/- standard deviation, decrease of log(10) CFU per milliliter of 3.65 +/- 1.02) compared with those of ampicillin alone (decrease of log(10) CFU per milliter of 0.07 +/- 0.8) and mecillinam alone (decrease of log(10) CFU per milliliter of 1.6 +/- 0.05) (P < 0.001). When bactericidal synergism can be demonstrated for mecillinam-ampicillin in vitro in a case of gram-negative-bacillary meningitis this combination may be useful in the therapy of the illness.