Although it is generally accepted that the acid/base ratio of tissue, as represented by the pH, is strictly regulated to maintain normal function, recent studies in the nervous system have shown that neuronal activity can result in significant shifts in pH. In the vertebrate retina, many cellular phenomena, including neuronal activity, are regulated by a circadian clock. We thus investigated whether a circadian clock regulates the pH of the retina. pH-sensitive microelectrodes were used to measure the extracellular pH of the in vitro goldfish retina superfused with a bicarbonate-based Ringer solution in the subjective day and night; that is, under conditions of constant darkness. These measurements demonstrated that a circadian clock regulates the pH of the vertebrate retina so that the pH is lower at night compared to the day. This day-night difference in retinal pH was observed at two different values of Ringer solution pH, indicating that the circadian phenomenon is independent of the superfusion conditions. The circadian-induced shift in pH was several times greater than light-induced pH changes and large enough to influence synaptic transmission between retinal neurons. These findings indicate that a circadian clock regulates the pH of the vertebrate retina. Thus, an intrinsic oscillator in neural tissue may modulate metabolic activity and pH as part of normal daily function.