The aminoglycosides are used clinically in combination with beta-lactam antibiotics. The combined use, however, produces an interaction and inactivation of the antibiotics. A study was designed to investigate the kinetics of the interaction in vitro. Four concentrations of aminoglycosides (5 to 20 micrograms of gentamicin and tobramycin per ml) and penicillins (100 to 600 micrograms of carbenicillin and ticarcillin per ml) were incubated in plasma (3 days, 37 degrees C). Samples taken at 12-h intervals were analyzed for both aminoglycosides (radioimmunoassay) and penicillin (high-pressure liquid chromatography). In controls, degradation of all four antibiotics were by first-order reactions. In incubation mixtures of two antibiotics, the rate of loss of the aminoglycosides was greater than that in the controls, whereas the rate of loss of penicillins was not significantly increased. The loss of penicillins in incubation mixtures still appeared to be by first-order reactions. However, semilogarithmic plots of aminoglycoside concentrations were curvilinear, suggesting a second-order reaction. Aminoglycoside concentrations in incubation mixtures were fitted by computer to a model incorporating a second-order interaction between aminoglycosides and penicillins and the first-order loss of penicillin from the mixture. The interaction rate constant averaged 2.2 X 10(-4) (micrograms/ml h)-1 for interaction of both carbenicillin and ticarcillin with gentamicin and 1.6 X 10(-4) (micrograms/ml h)-1 for interaction of the penicillins with tobramycin. The effect of the interaction in vivo was examined by computer simulation using the kinetic parameters determined in vitro.