Several laboratories have demonstrated the persistence of photosensitive rhythms related to melatonin secretion in cultured chick pineals. We describe here a system using dispersed chick pineal cells in static culture, which displays a rhythm of melatonin release for at least two weeks under cyclic lighting conditions, and for at least 4 cycles under constant red light. Using a rapid and specific extraction assay for the [14C]melatonin formed (from [14C]tryptophan) and secreted by these cells, we have examined the effects of perturbations (light, dark, and potassium) on the amplitude, period, and phase of the melatonin rhythm. The period in constant red light was close to 20 h, but in constant white light (or 12:12 cycles) it was closer to 24 h. Four-hour pulses of white light (in otherwise constant red light) caused an acute fall in melatonin output, and phase-dependent phase shifts of the rhythm relative to controls. Pulses of darkness (in otherwise constant red light) tended to increase melatonin output, and caused phase-dependent phase shifts. Elevated potassium concentrations increased melatonin output and the amplitude of the rhythm, but did not change the period. Four-hour pulses of low (5.4 mM) potassium (in otherwise constant high potassium) mimicked the acute effect of light, reducing melatonin output, but did not induce appreciable phase shifts. Changes in membrane potential appear more likely to be involved in the regulation of melatonin output (and thus be regulated by the pacemaker) than to be involved in regulation of the pacemaker which generates the melatonin rhythm.