Groups of 8-week-old chickens were killed at six time points in their light-dark cycle, and pineal glands were removed and assayed individually for N-acetyltransferase activity, hydroxyindole-O-methyltransferase activity, and melatonin content. The tenfold nocturnal rise in melatonin was phased identically with the 27-fold increase in N-acetyltransferase activity. The relatively small changes (20 percent) in hydroxyindole-O-methyltransferase activity did not appear important in causing the large changes in melatonin. The phase of the rhythms of N-acetyltransferase activity and melatonin content in chickens relative to the phase of the light-dark cycle was qualitatively similar to that of rats. In contrast, the sleep-wake cycle of chickens is about 180° out of phase with that of rats.