The reduction of chromium(VI) to Cr(III) results in the formation of reactive intermediates that contribute to the cytotoxicity, genotoxicity, and carcinogenicity of Cr(VI)-containing compounds. Previous studies suggest that human microsomal Cr(VI) reduction likely proceeds through cytochrome b(5). In order to better understand Cr(VI) toxicity in humans, the role of cytochrome b(5) in combination with P450 reductase was examined in the reductive transformation of Cr(VI). Proteoliposomes containing human recombinant cytochrome b(5) and P450 reductase were constructed. The ability of P450 reductase to mediate efficient electron transfer from NADPH to cytochrome b(5) was confirmed by spectral analysis. The NADPH-dependent Cr(VI) reduction rate mediated by proteoliposomes was then compared to that of human microsomes. When these rates were normalized to equivalent cytochrome b(5) concentrations, the NADPH-dependent Cr(VI) reduction rates mediated by human microsomes were essentially identical to those for proteoliposomes containing cytochrome b(5) plus P450 reductase. Proteoliposomes containing only P450 reductase or cytochrome b(5) exhibited poor Cr(VI) reducing capabilities. Since it had been previously shown that trace amounts of iron (Fe) could dramatically stimulate microsomal Cr(VI) reduction, the ability of Fe to stimulate Cr(VI) reduction by proteoliposomes was examined. Both ferric chloride (FeCl(3)) and ferric adenosine-5'-diphosphate (FeADP) were shown to stimulate Cr(VI) reduction; this stimulation could be abolished by the addition of deferoxamine, a specific Fe(III) chelator. The NADPH-dependent reduction rates of various ferric complexes by proteoliposomes were sufficient to account for the increased Cr(VI) reduction rates seen with the addition of FeCl(3) or FeADP. Cr(V) was detected by electron paramagnetic resonance (EPR) spectroscopy as a transient intermediate formed during NADPH-dependent Cr(VI) reduction mediated by proteoliposomes containing cytochrome b(5) and P450 reductase. Overall, cytochrome b(5) in combination with P450 reductase can account for the majority of the NADPH-dependent Cr(VI) reduction seen with human microsomes.