The in vitro inactivation of gentamicin, tobramycin, and netilmicin, when combined with carbenicillin, azlocillin, and mezlocillin, was studied. Plasma samples containing the aminoglycosides at a concentration of 5-8 micrograms/ml in combination with the penicillins in concentrations of 500, 250, and 50 micrograms/ml were incubated at room temperature and 37 degrees C for 0.3, 1, 3, 6, and 9 days. The aminoglycoside concentration was determined by radioimmunoassay or enzyme immunoassay. The extent of inactivation was dependent on penicillin concentration, contact time, and temperature. Penicillin concentrations of 500 micrograms/ml caused the greatest loss of aminoglycoside, while little loss occurred at the 50-micrograms/ml penicillin level. Carbenicillin, in concentrations of 250 and 500 micrograms/ml, inactivated all three aminoglycosides to a greater extent than either azlocillin or mezlocillin. The initial rate of decline in aminoglycoside concentration was greater at 37 degrees C than at room temperature. The new acylureidopenicillins, azlocillin and mezlocillin, inactivate the aminoglycosides studied, in a similar manner to that previously described for carbenicillin.