Pineal serotonin and melatonin levels and the activities of hydroxyindole-0-rnethyltransferase (HIOMT) and N-acetyltransferase (NAT) were studied over a 24-hour period in the pineal gland of the diurnally active Richardson’s ground squirrel ( Spermophilus richardsonii). Under alternating light-dark conditions (light:dark hours 14:10), pineal serotonin and melatonin levels exhibited a rhythm with high values occurring either during the day (serotonin) or during the night (melatonin). NAT activity was also markedly increased during darkness. HIOMT activity exhibited no 24-hour variation. Exposure of squirrels to constant light for 7 days exaggerated the serotonin rhythm, but obliterated the cycles of NAT and melatonin. Under constant darkness (for 7 days), the rhythms in serotonin, melatonin and NAT persisted, each having a period of about 24 h. In the second study, ground squirrels were exposed to light-dark cycles of either 8:16, 10:14 or 14:10. Under each of these photoperiodic environments, rhythms in pineal NAT and melatonin were apparent. Increasing the daily dark period from 10 to 14 h caused a prolongation of the elevated NAT and melatonin levels. However, a further prolongation of the daily dark period (to 16 h) did not further increase the duration of the rise in NAT and melatonin. The results show that continual light exposure (irradiance of 200 µW/cm<sup>2</sup>) for 7 days suppresses the pineal rhythms in both NAT activity and melatonin level in the Richardson’s ground squirrel. Conversely, light exposure, rather than depressing the serotonin rhythm, actually exaggerates it. Constant darkness for 7 days has little influence on the 24-hour rhythms of either NAT or melatonin. In the Richardson’s ground squirrel, it appears that the quantity of melatonin formed may be in part related to the duration of the daily dark period.