Localized, subsarcolemmal Ca2+ release (LCR) via ryanodine receptors (RyRs) during diastolic depolarization of sinoatrial nodal cells augments the terminal depolarization rate. We determined whether LCRs in rabbit sinoatrial nodal cells require the concurrent membrane depolarization, or are intrinsically rhythmic, and whether rhythmicity is linked to the spontaneous cycle length. Confocal linescan images revealed persistent LCRs both in saponin-permeabilized cells and in spontaneously beating cells acutely voltage-clamped at the maximum diastolic potential. During the initial stage of voltage clamp, the LCR spatiotemporal characteristics did not differ from those in spontaneously beating cells, or in permeabilized cells bathed in 150 nmol/L Ca2+. The period of persistent rhythmic LCRs during voltage clamp was slightly less than the spontaneous cycle length before voltage clamp. In spontaneously beating cells, in both transient and steady states, LCR period was highly correlated with the spontaneous cycle length; and regardless of the cycle length, LCRs occurred predominantly at a constant time, ie, 80% to 90% of the cycle length. Numerical model simulations incorporating LCRs reproduce the experimental results. We conclude that diastolic LCRs reflect rhythmic intracellular Ca2+ cycling that does not require the concomitant membrane depolarization, and that LCR periodicity is closely linked to the spontaneous cycle length. Thus, the biological clock of sinoatrial nodal pacemaker cells, like that of many other rhythmic functions occurring throughout nature, involves an intracellular Ca2+ rhythm.