We previously reported findings that NADPH/cytochrome P450 reductase can generate superoxide anion radical (O2*-) from heterocyclic amines (HCA) and from many anticancer agents in vitro. Here we present more evidence in which O2*- is generated when recombinant human cytochrome b5 reductase (rh-Cytb5Rd) was incubated with HCAs such as IQ and MeIQ in the presence of NADH in vitro. This indicates that free radical generation by rh-Cytb5Rd in the presence of HCA may add new insight into the damage of DNA in addition to the previously known mechanism: interaction of activated HCA-intermediates to form DNA adduct.