The chemiluminescent reaction of lucigenin with various biological substances has been studied. Chemiluminescence of lucigenin is produced by the addition of either hydrogen peroxide or organic reducing compounds to lucigenin in an alkaline solution. On the basis of these reaction, we have developed highly sensitive chemiluminescent methods for the detection of enzyme immunoassay, especially using alkaline phosphatase as a label enzyme, and also for HPLC of corticosteroids or p-nitrophenacyl esters of carboxylic acids. The detection limits of enzymes were 10^-19-10^-20 mol per assay, corticosteroids and p-nitrophenacyl esters were 500 fmol per injection.