The key regulatory enzyme in melatonin synthesis is arylalkylamine N-acetyltransferase (AANAT). In vivo, AANAT activity in chicken retinal photoreceptor cells exhibits a circadian rhythm that peaks at night. The purpose of the present study was to investigate the temporal development of light/dark and circadian oscillations of AANAT activity in cultured retinal cells prepared from 6- and 8-day-old chicken embryos (E6, E8, respectively). Photoreceptor cells prepared from E6 retinas and incubated under a 14-h light/10-h dark (LD) cycle of illumination for 5-7 days displayed prominent daily fluctuations in AANAT activity on days 5 and 6 in vitro. However, when E6 cells, incubated for 5 days under LD, were transferred to continuous (24 h/day) darkness (DD) on day 6, no daily pattern of activity was observed. This result indicates that AANAT fluctuations were light-driven and not circadian at this stage. In contrast, cells prepared from E8 embryos and incubated under conditions identical to those for E6 cells displayed prominent rhythms of AANAT activity in both LD and DD, indicative of circadian control. To determine if circadian control of AANAT activity would develop in E6 cells incubated for a longer period of time to allow maturation, cells were incubated for 8 days in LD followed by 2 days in DD. AANAT activity in these cells was rhythmic in both LD and DD. In cells incubated in this manner, a 2-h light pulse in the middle of the subjective night suppressed AANAT activity, indicating that the enzyme activity in the cultured cells is acutely suppressed by light, as it is in vivo. These results indicate that the ability to express circadian regulation of AANAT activity is an intrinsic property of retinal cells that can develop in vitro. Development of light-dark regulation of AANAT activity appears to precede the circadian clock-control of enzyme activity.