This paper reports a non equilibrium competitive enzyme immunoassay method using enzyme-labeled antibodies, for the quantitation of melatonin in chloroform-extracted samples. Its principle is as follows: methoxytryptamine hemisuccinate-human serum albumin conjugate physically absorbed onto a polystyrene sphere and melatonin to be measured, compete for a limited and fixed amount of peroxidase-labeled anti melatonin IgG. After incubation and washings the enzymatic activity bound to the sphere was measured with a chromogenic substrate. This simple method can detect as low as 22 fm of melatonin and is fairly precise. We also present its application to the determination of melatonin in serum and pineal gland.