The administration of gentamicin at least 1 hour before administration of ampicillin in neonates has been advocated because of in vitro inactivation of aminoglycosides by beta-lactam antibiotics. This method would cause a delay in ampicillin dosing in the treatment of serious bacterial infections and unnecessarily complicate nursing procedures. We studied the effect of varying concentrations of ampicillin (50, 100, 200, and 400 micrograms/ml) on aminoglycosidic antibiotics in vitro with the use of stock solutions diluted in pooled sera obtained from cord blood and incubated samples at 25 degrees C, 37 degrees C, and 40 degrees C. We found inactivation of aminoglycosides to be dependent on time, temperature, and ampicillin concentration, but the degree of inactivation was small and does not support temporal separation of parenteral administration of ampicillin and aminoglycosides to neonates.