Evidence is presented that the substrate for the drug oxidizing system, NADPH, binds to zinc ions. Zinc ions bind selectively to NADPH but not to NADH. By using equilibrium gel filtration and acid titrations of Zn2+ and NADPH, the molar ratio of metal to nucleotide was determined at 2 : 1, as well as the formation constant of 10(6.75). Results from 31P-nuclear magnetic resonance (NMR), ultraviolet and fluorescence spectra of the complex indicated the possible binding sites of zinc to NADPH. In previous studies we have shown that zinc ions inhibit the metabolism of drugs by mixed function oxidases in liver microsomes. The formation of the Zn2+-NADPH complex suggests the mechanism by which zinc ions may inhibit the drug oxidizing system.