The reduction of nitrofurantoin by purified liver nitrofurantoin reductase was followed by the production of superoxide radicals, which were detected by the reduction of epinephrine. The conditions for the formation of superoxide radicals were optimized. The maximum superoxide radical formation occurred at approximately 3.5 mu g purified reductase, with optimum pH of 7.8 and at 0.05 mM nitrofurantoin concentration. The K-m and V-max values were calculated as 1.95 x 10(-2) mM and 4.81 nmol superoxide formed per minute. An obliteration of the banding pattern was observed on the agarose gel electrophoresis of sheep liver DNA, which was incubated in the nitrofurantoin-nitrofurantoin reductase system, as a possible consequence of the generation of superoxide radicals during the reduction of nitrofurantoin by nitrofurantoin reductase.