The activity of N-acetyltransferase in theepiphysis is significantly increased by 3 h of darkness after the evening switching off of the animal room lights, but not by a similar period of darkness experienced during daylight hours. However, in rats maintained in constant light, the enzyme activity increases after 3 h of darkness experienced during daylight hours. It would appear that the increase in enzyme activity is a complex function of both darkness per se and a rhythmic ability of the organism to respond to darkness. Administration of the monoamine oxidase inhibitor pargyline during the day produces first a decrease in enzyme activity and then an increase by a factor of 4 to 8. The degree of the increase is not dependent upon the time of day at which pargyline is given. Administration of pargyline in the evening causes an increase in N-acetyltransferase activity by a factor of only 2 to 3 and does not prevent the decrease in activity caused by turning the lights on at night. The administration of pargyline does not permit rats to respond to darkness during the day by increased N-acetyltransferase activity.