Serotonin N-acetyltransferase (NAT) activity in chicken pineal homogenates is increased 16-fold in the presence of high-molarity phosphate buffer (0.35 M) as compared with its activity in low-molarity (0.05 M) phosphate buffer. This phosphate effect on NAT does not depend on ionic, osmotic, or pH changes; rather, it appears to be a direct effect of phosphate on NAT activity. Phosphate also stabilizes NAT activity to thermal inactivation and inactivation caused by incubation at 4 degrees C for 48 h. Stimulation of NAT activity by phosphate occurs only in chick pineal and retina, not in chick cerebrum, cerebellum or liver, nor in rat pineal or other tissues tested. There is a correlation between the occurrence of the phosphate effect and the occurrence of endogenous NAT circadian rhythmicity and light inactivation. The effect of phosphate on NAT activity in homogenates may reflect physiological mechanisms of NAT regulation.