A radioimmunoassay for the determination of concentrations of melatonin in the plasma of rhesus monkeys has been developed. Antiserum for the assay was raised against N-acetyl serotonin and there was a 100% cross-reaction with melatonin. Cross-reactivity with closely related indoles, precursors and metabolites was less than or equal to 1%. The lower limit of sensitivity of the assay was 4 pg/tube. The assay has been used for the investigation of diurnal variations and cyclical changes in melatonin concentrations in peripheral plasma of the rhesus monkey. The concentrations of melatonin ranged between 26.6 and 85.3 pg/ml during sampling for 24 hr. There was a distinct diurnal variation in the concentration of melatonin in plasma. The concentration during darkness (61.0 ± 7.1 (S.E.M.) pg/ml) was greater (P < 0.01) than that during illumination (40.1 ± 6.1 pg/ml). There were no significant differences in the concentration of melatonin in plasma at any time during the 28 day menstrual cycle of the rhesus monkey.