Radical formation during autooxidation of the polyene antibiotic amphotericin B was monitored by following the kinetics of decay of the ESR signal of a stable nitroxide. The kinetics were seen to depend both on antibiotic and on nitroxide concentration. The radicals formed were studied by spin trapping. Three preparations--clinical Fungizone, amphotericin B suspended in buffer, and amphotericin B in buffer - 10% dimethyl sulfoxide--yielded spin adducts of different nature and/or concentrations. In the absence of dimethyl sulfoxide, amphotericin B yielded at least two carbon-centered radicals whose spectra indicated restricted mobility. This suggests that the radicals arise from the whole amphotericin B molecule located in molecular aggregates present in the preparations, and not from smaller and possibly more water-soluble fragments of the antibiotic. In the presence of dimethyl sulfoxide the spin adducts derived from secondary carbon radicals originated from this solvent. Their spectra were indicative of fast tumbling. Direct evidence for autooxidation was obtained by measuring oxygen consumption. All processes examined occurred at the same time scale observed for drug inactivation, in agreement with the idea that loss of activity is due to antibiotic autooxidation.