The efficacy of cyclacillin as an antimicrobial agent against Escherichia coli was assessed in vivo in mice infected with low numbers of bacteria and compared to the relative effectiveness of the antibiotic against the same organisms in vitro. Treatment of mice with cyclacillin resulted in a rapid clearance of E. coli from the blood and their greater killing in the spleen and liver. Furthermore, a significantly higher antibody plaque response against E. coli developed in cyclacillin-treated mice than in untreated mice or in those given ampicillin. The increased immunogenicity of the E. coli in the antibiotic-treated mice appeared to be due to high levels of cyclacillin in the animals and rapid killing of the bacteria in vivo. In vitro experiments showed that injection of normal mouse peritoneal exudate macrophages which had been incubated with E. coli together with cyclacillin resulted in a greater immunogenicity of the bacteria than when the injection mixture was composed of E. coli which had been incubated alone or only with macrophages. These results suggest that the in vivo effectiveness of an antibiotic such as cyclacillin against a gram-negative organism such as E. coli may be due in part to an effect on the immunogenicity of the bacteria.