Adult male albino rats were acclimated to constant light (light:dark-LD-24:0) or to darkness interrupted with brief periods of light at 6 h intervals (LD 1/4:5 3/4 X 4) concurrently with rats maintained in a LD 14:10 photoperiodic cycle. The activity and rhythmicity of pineal serotonin N-acetyltransferase (NAT) was examined at regular intervals for 24 hours in rats maintained in the experimental photoperiods and compared to pineal NAT activity and rhythmicity in rats maintained in the LD 14:10 photoperiod. The results indicate that constant light is capable of depressing nocturnal levels of rat pineal NAT and obliterating the pineal NAT rhythm. Likewise, rats subjected to darkness interrupted with brief periods of light at 6 h intervals experienced a similar response in pineal NAT activity to animals subjected to constant light, i.e., pineal NAT activity was persistently low and the rhythmicity was obliterated. The results are discussed relative to the hypothesis that the pineal NAT activity responds to an endogenous rhythm in photoperiodic time measurement. The evidence herein suggests that the time of occurrence of environmental light in the photoperiod is more important in determining pineal NAT activity and/or rhythmicity than is the total amount of darkness or the dark to light ratio to which animals may be subjected.