This study was undertaken to compare the susceptibility to inactivation of isepamicin with amikacin and gentamicin when exposed to different beta-lactams, beta-lactamase inhibitors, and heparin. The aminoglycosides (5, 10, 20, and 50 micrograms/ml) were incubated in human serum with ampicillin, azlocillin, aztreonam, carbenicillin, ceftazidime, piperacillin, and ticarcillin (100 and 600 micrograms/ml) and with clavulanate, cilastatin, 1:1 imipenemcilastatin, oxacillin, and sulbactam (20 and 120 micrograms/ml) for 48 h at 37 degrees C. Aminoglycoside concentrations were measured by fluorescence polarization immunoassay (FPI) after 0, 8, and 48 h of incubation and by radial diffusion bioassay after 48 h of incubation. Each of the three aminoglycosides was also added to whole blood containing either heparin (100 U/ml) or 0.5% EDTA as a control and assayed after 6 h by FPI. The degree of inactivation of isepamicin by the beta-lactams was significantly less than that by amikacin (P less than 0.003) and gentamicin (P less than 0.0002) when determined by bioassay. Piperacillin, carbenicillin, and azlocillin produced the greatest amount of inactivation, and cilastatin and oxacillin produced the least. A similar pattern was observed when the degree of inactivation was measured by FPI. A significant difference in the degree of inactivation was noted between isepamicin and gentamicin (P less than 0.003 at 8 h and P less than 0.006 at 48 h) but not between isepamicin and amikacin (P greater than 0.7 at 8 h and P greater than 0.08 at 48 h). Aminoglycoside determinations by FPI were not influenced by the presence of heparin. In summary, isepamicin was found to be at least as stable as amikacin against inactivation by beta-lactam compounds and beta-lactamase inhibitors. Heparin (100 U/ml) did not influence aminoglycoside determinations by FPI.