Abstract: The chemiluminescence reaction of luminol with reducing agents was studied by a flow-injection analysis. We found that reducing agents directly produce a chemiluminescence with luminol in the presence of hexacyanoferrate(III) and hexacyanoferrate(II) as a catalyst in an alkaline solution. To explain the chemiluminescence of luminol with reducing agents, ESR measurements were performed. ESR showed that superoxide anion radicals were generated from uric acid, dihydroxyacetone, hydroxylamine, cortisone and glucose in an alkaline solution by using 5,5'-dimethyl-1-pyrroline N-oxide (DMPO) as a spin-trap agent. The ascorbate radical was observed for ascorbic acid without DMPO. For phenacyl alcohol, a new radical without DMPO was observed. It is reported that the superoxide anion radical is a crucial intermediate in luminol chemiluminescence and that the chemiluminescence is generated in the presence of free radicals. Therefore, it was suggested that these radicals resulted from the reducing agents in the alkaline solution participate in luminol chemiluminescence. The detection limits (S/N=3) of uric acid, phenacyl alcohol, hydroxylamine, ascorbic acid, dihydroxyacetone, cortisone and glucose were 40, 60, 70, 200, 430, 440 and 75 ng, respectively.