1. The dose dependence of the cholinergic agonist acetylcholine (ACh) and the beta-adrenergic agonist isoprenaline (Iso) were determined for the hyperpolarization-activated current (If) and the L-type Ca2+ current (ICa,L) in single cells isolated from the rabbit sino-atrial (SA) node. 2. ACh inhibited If by a negative shift of its activation curve with a maximal effect of -9.9 mV; half-maximal effect was produced by 0.019 microM ACh. High ACh concentrations were required to inhibit ICa,L only partially (31% inhibition at 300 microM). 3. In contrast, If and ICa,L responded to Iso over a similar dose range, with concentrations for half-maximal enhancement of 0.0136 and 0.0070 microM, respectively. 4. The effects on spontaneous activity of ACh (range 0.001-0.03 microM) and Iso (range 0.001-1 microM) were investigated. ACh decreased the slope of diastolic depolarization at concentrations similar to those inhibiting If (> 50% at 0.03 microM). Iso enhanced diastolic depolarization at concentrations similar to those affecting both If and ICa,L (half-maximal effect at 0.027 microM). 5. In a ramp-clamp protocol simulating diastolic depolarization, the threshold for activation of inward nifedipine-sensitive current was -41.22 +/- 0.68 mV. Although enhancing ICa,L, Iso did not affect this threshold. 6. Half-maximal ACh concentrations for inhibition of automaticity and If are similar and are lower than the threshold concentrations for modulation of ICa,L; this argues against a role of ICa,L in direct muscarinic modulation of pacemaking. In contrast, modulation of If, ICa,L and automaticity occur at similar Iso concentrations. The difference between maximum diastolic potential (-61.95 +/- 0.93 mV) and the threshold for Iso-stimulated ICa,L (-39.54 +/- 1.03 mV) suggests that this current plays a role only at later stages of diastolic depolarization.