There is reason to believe that resting free calcium concentration [Ca2+]i in neurons in the suprachiasmatic nucleus (SCN) may vary with the circadian cycle. In order to start to examine this hypothesis, optical techniques were utilized to estimate resting Ca2+ levels in SCN cells in a rat brain slice preparation. [Ca2+]i measured from the soma was significantly higher in the day than in the night. Animals from a reversed light-dark cycle were used to confirm that the phase of the rhythm was determined by the prior light-dark cycle. The rhythm in Ca2+ levels continued to be expressed in tissue collected from animals maintained in constant darkness, thus confirming the endogenous nature of this variation. Interestingly, the rhythm in Ca2+ levels was not observed when animals were housed in constant light. Finally, the rhythm in Ca2+ levels was prevented when slices were exposed to tetrodotoxin (TTX), a blocker of voltage-sensitive sodium channels. Similar results were obtained with the voltage-sensitive Ca2+ channel blocker methoxyverapamil. These observations suggest a critical role for membrane events in driving the observed rhythm in Ca2+. Conceptually, this rhythm can be thought of as an output of the circadian oscillator. Because [Ca2+]i is known to play a critical role in many cellular processes, the presence of this rhythm is likely to have many implications for the cell biology of SCN neurons.