The response of the circadian rhythm in N-acetyltransferase (NAT) activity to phase-shifted light cycles was examined in vitro in explant cultures of chick (Gallus domesticus) pineal glands. Bisected portions of glands, obtained from birds housed in a light-dark cycle (LD 12:12), were explanted into culture and maintained under one of three light cycles (LD 12:12), the phase of which was either 1) similar to that of the birds' previous cycle, 2) seven hours phase-delayed, or 3) six hours phase-advanced. Following two to three days of exposure to the respective light cycles, cultures were placed into continuous darkness (DD). Sampling from cultures during exposure to DD revealed a circadian rhythm of NAT activity. In each case, the phase of the subsequent rhythm of enzyme activity in DD reflected that of the preceding in vitro light cycle. A distinct phase difference of approximately 180 degrees was observed between cultures exposed to opposite lighting regimes. These results indicate entrainment of the circadian rhythm of NAT activity to the prevailing in vitro light cycle and suggest a direct interaction between pineal photoreception and the circadian "clock" controlling NAT.