The extracellular pH of living tissue in the retina and elsewhere in the brain is lower than the pH of the surrounding milieu. We have also shown that the pH gradient between the in vitro retina and the superfusion solution is regulated by a circadian (24-hr) clock so that it is smaller in the subjective day than in the subjective night. We show here that the circadian changes in retinal pH result from a clock-mediated change in the generation of H⁺ that accompanies energy production. To demonstrate this, we suppressed energy metabolism and recorded the resultant reduction in the pH difference between the retina and superfusate. The magnitude of the reduction in the pH gradient correlated with the extent of energy metabolism suppression. We also examined whether the circadian-induced increase in acid production during the subjective night results from an increase in energy metabolism or from the selective activation of glycolysis, compared to oxidative phosphorylation. We found that the selective suppression of either oxidative phosphorylation or glycolysis had almost identical effects on the dynamics and extent of H⁺ production during the subjective day and night. Thus, the proportion of glycolysis and oxidative phosphorylation is maintained the same regardless of circadian time, and the pH difference between the tissue and superfusion solution can therefore be used to evaluate total energy production. We conclude that circadian clock regulation of retinal pH reflects circadian regulation of retinal energy metabolism.