Block of the hyperpolarization-activated "pacemaker" current (i(f)) by the bradycardic agent UL-FS 49 was studied in isolated sino-atrial (SA) node myocytes. Using repetitive activation/deactivation protocols, micromolar concentrations of UL-FS 49 blocked i(f) in a dose-dependent fashion. Block development was slow, with time constants decreasing with drug concentration and ranging from 25.8 s at 10 microM to 75.5 s at 1 microM UL-FS 49. Block did not develop in cells held at -35 mV, at which voltage i(f) channels are closed, indicating that channels must open before blocking occurs. Apparently in contrast with the requirement of negative voltages for block development, block was relieved by hyperpolarization with a time course slower than current kinetics. Due to the hyperpolarization-induced block relief, current/voltage (I/V) relations in the presence of UL-FS 49 displayed inward-going rectification. Experimental data fitted the hypothesis that UL-FS 49 behaves as an "open channel" blocker of "single-ion" i(f) channels. Block occurs within the pore, at a distance of about 39% of the membrane thickness from its internal side.