Pineal weight and serotonin (5-HT) and norepinephrine (NE) contents were studied in male Sprague-Dawley rats that were maintained under controlled light:dark conditions (LD 14:10; lights on 0700-2100) and that received daily subcutaneous injections of either melatonin (20 micrograms in 0.1 ml per animal) or the same volume (0.1 ml) of vehicle alone, at one of two times (0800-0900 or 1800-1900). Animals were sacrificied at four times (1000, 1400, 2000, or 2300) on the day after the last of the 7 consecutive d of injection. Pineal glands were quickly weighed and then frozen for 5-HT and NE assay by the Maickel and Miller extraction and fluorescence methods. Pineal NE content showed differences related to time of day, in confirmation of early work. But no effects attributable specifically to melatonin were found. Melatonin also failed to affect pineal 5-HT content significantly. But injection of either melatonin or vehicle at 1800-1900 led to a reduction in 5-HT content averaging 36% when sampled at either 2000 or 2300, and in comparison with animals injected at 0800-0900. It is suggested that a stresslike or zeitgeberlike effect of injections within a critical period at the end of the daily light phase can cause an earlier-than-normal daily fall in pineal 5-HT content.