Clinical microbiology laboratory experiences wherein aminoglycoside serum assay results appeared inconsistent with the amount of the compound given when administered in combination with a semisynthetic penicillin led us to compare the interaction of three currently available aminoglycoside antibiotics with carbenicillin, as a representative semisynthetic penicillin given in large doses, and with piperacillin, a new broad-spectrum semisynthetic penicillin. Amikacin, gentamicin, and tobramycin were incubated in serum in vitro with various concentrations of carbenicillin and piperacillin. In the presence of very high concentrations of the two penicillins, tobramycin was the most rapidly inactivated aminoglycoside, gentamicin was next, and amikacin was only slightly inactivated. With low concentrations of the two penicillins, the rates of aminoglycoside inactivation were negligible. Carbenicillin, in high concentration, inactivated the aminoglycosides more rapidly than piperacillin. In patients experiencing renal failure, the maintenance of moderate serum levels of carbenicillin and piperacillin may be important in attempting to maintain adequate aminoglycoside serum levels.