Circadian clocks are intracellular molecular mechanisms that allow the cell to anticipate the time of day. We have previously reported that the intact rat heart expresses the major components of the circadian clock, of which its rhythmic expression in vivo is consistent with the operation of a fully functional clock mechanism. The present study exposes oscillations of circadian clock genes [brain and arylhydrocarbon receptor nuclear translocator-like protein 1 (bmal1), reverse strand of the c-erba␣ gene (rev-erba␣), period 2 (per2), albumin D-element binding protein (dbp)] for isolated adult rat cardiomyocytes in culture. Acute (2 h) and/or chronic (continuous) treatment of cardiomyocytes with FCS (50% and 2.5%, respectively) results in rhythmic expression of circadian clock genes with periodicities of 20 -24 h. In contrast, cardiomyocytes cultured in the absence of serum exhibit dramatically dampened oscillations in bmal1 and dbp only. Zeitgebers (timekeepers) are factors that influence the timing of the circadian clock. Glucose, which has been previously shown to reactivate circadian clock gene oscillations in fibroblasts, has no effect on the expression of circadian clock genes in adult rat cardiomyocytes, either in the absence or presence of serum. Exposure of adult rat cardiomyocytes to the sympathetic neurotransmitter norephinephrine (10 M) for 2 h reinitiates rhythmic expression of circadian clock genes in a serumindependent manner. Oscillations in circadian clock genes were associated with 24-h oscillations in the metabolic genes pyruvate dehydrogenase kinase 4 (pdk4) and uncoupling protein 3 (ucp3). In conclusion, these data suggest that the circadian clock operates within the myocytes of the heart and that this molecular mechanism persists under standard cell culture conditions (i.e., 2.5% serum). Furthermore, our data suggest that norepinephrine, unlike glucose, influences the timing of the circadian clock within the heart and that the circadian clock may be a novel mechanism regulating myocardial metabolism.heart; metabolism; neurohumoral; rat; zeitgebers CIRCADIAN CLOCKS are controlled by a set of genes that generate self-sustained positive and negative transcriptional feedback loops with a free-running period of 24 h (7,11,40). This molecular mechanism is intrinsic to the cell, persisting in cultured cells such as fibroblasts, vascular smooth muscle cells, and various cell lines (3,13,20,23). Circadian clocks confer the selective advantage of anticipation, conditioning the cell to changes in its environment, such that it can respond rapidly to a specific extracellular signal at an appropriate time of the day. For example, our laboratory (30) has recently hypothesized that the circadian clock within cardiac and skeletal muscle allows the myocytes to anticipate diurnal variations in circulating fatty acids through modulation of fatty acid oxidative capacity at a transcriptional level. However, the existence of an intracellular circadian clock mechanism that is intrinsic to either the cardiomyocyte or...