A novel and time-saving method for assessing bactericidal activities of beta-lactam antibiotics on agar plates is described. Minimal inhibitory concentrations (MICs) were determined by the agar dilution method. A potent beta-lactamase solution was sprayed onto the plates to inactivate the antibiotic. After further incubation at 37 degrees C overnight, the minimal concentration at which no visible growth occurred on the plates was defined as minimal bactericidal concentration (MBC). With undiluted culture as the inoculum, strains of Staphylococcus aureus, Escherichia coli, and Klebsiella pneumoniae showed a marked increase in MBC values compared with the values of MIC. There was a marked decrease in both the MICs and MBCs with diminution of inoculum size. The two concentrations were almost the same when the inoculum size was decreased to a 10(-4) dilution. In contrast, MIC and MBC for enterococci showed no marked decrease with decrease in inoculum size. Although the present study was performed with beta-lactamase-unstable penicillins and cephalosporins, the method can be applicable with any beta-lactam antibiotic if optimal and potent enzymes are available.