The levels of N-acetylserotonin and melatonin (n-acetyl-5-methoxytryptamine) in the rat brain were studied. Male rats were housed under a photoperiod of 12L:12D. After 2 weeks, the rats were sacrificed in light or in dark at 4-hour intervals round the clock. Brain melatonin and N-acetylserotonin were extracted by chloroform at greater than pH 10 and ethyl acetate at less than pH 3 respectively and quantified by radioimmunoassay. There was a circadian rhythm of brain melatonin (F less than 0.05) with a nadir at 1600 h and a zenith at 0400 h. Brain N-acetylserotonin, unlike brain melatonin, demonstrated no diurnal variation. Effects of pinealectomy on brain N-acetylserotonin and melatonin were also studied. Male rats were housed as noted. After 2 weeks, they were pinealectomized or shamoperated. The animals were killed in light or in dark 1 week after operation. The levels of brain melatonin at 2400 h and 0400 h were significantly lowered (P less than 0.05) following pineal removal. Pinealectomy, however, has no observable effect on the level of brain N-acetylserotonin. Our findings suggest that 1) there is a diurnal rhythm of brain melatonin, 2) the elevation of brain melatonin in the dark period is dependent on the pineal, 3) brain N-acetylserotonin has no diurnal variation, and 4) brain N-acetylserotonin is not affected by pinealectomy.