The effects of sphingosine 1-phosphate (S-1-P) on pacemaker activity and underlying membrane currents were studied in isolated rabbit sino-atrial (SA) node cells. S-1-P (0.1 microM) reversibly increased the cycle length of spontaneous pacemaker activity from 560 to 1434 ms, and hyperpolarized the maximal diastolic potential (MDP) from -67 to -70 mV. In voltage-clamp experiments, S-1-P (1 microM) activated a pertussis toxin-sensitive, inwardly-rectifying, time-independent K+ current (I(K,ACh)) that had a reversal potential of -88 mV (K+ equilibrium potential -86 mV). S-1-P (1 microM) had no measurable effect on the L-type Ca2+ current (I(Ca,L)) or the hyperpolarization-activated inward current (I(f)) under basal conditions. In the presence of the beta-adrenergic agonist, isoproterenol (ISO, 0.1 microM), S-1-P (1 microM) reversed the ISO-induced increase in pacing rate, hyperpolarized the MDP and decreased the ISO-induced enhancement of both I(Ca,L) (from 171 to 118% of control) and I(f) (from 211 to 135% of control). These results demonstrate that under basal conditions S-1-P can significantly slow spontaneous pacing in rabbit SA node cells mainly due to activation of a background, inwardly-rectifying K+ current. In the presence of ISO, S-1-P also slows the spontaneous pacing rate due to activation of the same K+ current, as well as inhibition of I(Ca,L) and I(f).