Lipoyl group determination by lipoamide dehydrogenase (NADH: lipoamide oxidoreductase; EC 1.8.1.4) was examined using lipoyl lysine as a substrate. The reaction was monitored by the coupled oxidation of NADH at 340 nm absorbance. Dehydrogenase-mediated NADH oxidation was too slow to be used for the quantification of lipoyl groups in the concentration range 1 to 10 microM. However, when glutathione disulfide (GSSG) was added to the reaction mixture to regenerate the oxidized substrate for the enzyme, NADH oxidation was markedly enhanced. This GSSG-dependent enhancement of NADH oxidation was strongly dependent upon the lipoyl substrate, but was only slightly dependent on the amounts of GSSG without the substrate. In the presence of excess GSSG, NADH oxidation was linearly correlated to the concentration of lipoyl lysine up to 10 microM; this assay is suitable for determining micromolar concentrations of the lipoyl moiety.