1. The present study was undertaken to examine the responses of the sustained inward current (I(st)) to beta-adrenergic and muscarinic agonists in guinea-pig sino-atrial (SA) node cells using the whole-cell patch-clamp technique. I(st) was detected as the nicardipine (1 microM)-sensitive inward current at potentials between approximately -80 and +20 mV in the presence of low concentration (0.1 mM) of extracellular Ca2+, where the L-type Ca2+ current (I(Ca,L)) was practically abolished. 2. Beta-adrenergic agonist isoprenaline (ISO) in nanomolar concentrations not only increased the amplitude of I(st) but also shifted the membrane potential producing the peak amplitude (Vpeak) to a negative direction by approximately 15 mV without appreciably affecting potential range for the current activation. The stimulatory effect of ISO was concentration-dependent with an EC50 of 2.26 nM and the maximal effect (96.4+/-22.9% increase, n=6) was obtained at 100 nM ISO, when evaluated by the responses at -50 mV. 3. Bath application of acetylcholine (ACh) significantly inhibited I(st), which had been maximally augmented by 100 nM ISO; this inhibitory effect of ACh was concentration-dependent with an IC50 of 133.9 nM. High concentration (1000 nM) of ACh depressed basal I(st) by 10.5+/-2.0% (n=3). 4. In action potential clamp experiments, I(st) was also detected under control conditions and was markedly potentiated by exposure to ISO. 5. These results strongly suggest that I(st) not only contributes to the spontaneous action potentials of mammalian SA node cells but also plays a substantial role in mediating autonomic regulation of SA node pacemaker activity.