Kinetic studies of ampicillin action were made on exponentially growing Escherichia coli and on E. coli-spheroplasts using a range of inhibitory and subinhibitory concentrations of ampicillin. For each concentration, the value (ko--ka) representing the difference in generation rates of ampicillin-free culture (ko) and the generation rate of the culture with ampicillin (ka) was calculated and plotted against ampicillin concentration. A straight line relation was obtained with E. coli cells, its intersection with the abscissa, where (ko--ka) = o, give the concentration of ampicillin which exerts no inhibitory action on the cells (0.25 microgram/ml). When ka was plotted against ampicillin concentration, the relation was also linear. Its intersection with the abscissa gives the minimum lethal concentration of ampicillin on the bacterial cells (1.05 microgram/ml). With E. coli-spheroplasts such plots were non-linear which means that a different order of reaction was involved. This difference is probably due to a different mechanism of ampicillin action as revealed by the electroscanning microscopy.