The present study demonstrates that the biosynthesis of phospholipids in the inner nuclear layer cells of the chicken retina displays daily rhythms under constant illumination conditions. The vertebrate retina contains circadian oscillators and photoreceptors (PRCs) that temporally regulate its own physiology and synchronize the whole organism to the daily environmental changes. We have previously reported that chicken photoreceptors and retinal ganglion cells (RGCs) present significant daily variations in their phospholipid biosynthesis under constant illumination conditions. Herein, we demonstrate that cell preparations highly enriched in inner nuclear layer cells also exhibit a circadian-regulated phospholipid labeling after the in vivo administration of [(32)P]phosphate or [(3)H]glycerol both in animals maintained under constant darkness or light for at least 48h. In constant darkness, there was a significant incorporation of both precursors into phospholipids with the highest levels of labeling around midday and dusk. In constant light, the labeling of (32)P-phospholipids was also significantly higher during the day and early night whereas the incorporation of [(3)H]glycerol into phospholipids, that indicates de novo biosynthesis, was greater during the day but probably reflecting a higher precursor availability at those phases. We also measured the in vitro activity of phosphatidate phosphohydrolase and diacylglycerol lipase in preparations obtained from the dark condition. The two enzymes exhibited the highest activity levels late in the day. When we assessed the in vitro incorporation of [(14)C]oleate into different lysophospholipids from samples collected at different phases in constant darkness, reaction catalyzed by lysophospholipid acyltransferases II, labeling showed a complex pattern of daily activity. Taken together, these results demonstrate that the biosynthesis of phospholipids in cells of the chicken retinal inner nuclear layer exhibits a daily rhythmicity under constant illumination conditions, which is controlled by a circadian clock.