[U-14C]Benzidine and unlabeled benzidine were used to synthesize benzidinediimine for which n.m.r., i.r. and u.v./visible spectra were obtained. Examination of benzidinediimine in acetate pH 4 buffer by electron spin resonance spectroscopy revealed that a free radical cation of benzidine was produced with maximum radical concentration occurring in 3-4 min. In contrast, u.v./visible spectroscopy revealed the production of a charge-transfer complex with maximum concentration occurring in 6-7 min. Liquid chromatography with electrical and radiochemical detection indicated that benzidine was the final product of benzidinediimine reduction in acetate buffer. The rate of reduction was greater at higher pH's and the time course for production of the free radical and charge-transfer complex did not coincide at any pH examined. Addition of DNA to [14C]benzidinediimine resulted in DNA labeling which was completely inhibited by glutathione. These data suggest that charge-transfer complex formation neither precedes nor coincides with free radical formation. They are consistent with the hypothesis that the free radical cation observed during peroxidatic oxidation of benzidine is derived from one-electron oxidation.