The anti-anginal agent bepridil blocks slow Ca2+ channels in a variety of tissues. Since bepridil accumulates inside cells, the possibility exists that bepridil acts, at least partially, from inside the cell. To test this possibility, we examined the effects of a quaternary ammonium analog of bepridil, methylated bepridil, which presumably would enter the cells less readily, on the Ca2+-dependent slow action potentials of guinea pig papillary muscles (in 25 mM [K+]0) and rabbit pulmonary arteries (in tetraethylammonium chloride). In cardiac muscle, methylated bepridil had little effect on the slow action potentials at low stimulation frequencies (0.5 Hz), but at higher frequencies (1.0 and 2.0 Hz) the slow action potentials were depressed and/or the muscle was unable to follow each stimulation. These effects are similar to those obtained with bepridil, but bepridil was more potent than methylated bepridil. In vascular smooth muscle cells, methylated bepridil inhibited the slow action potentials at a somewhat lower dose than bepridil. We conclude that, in cardiac muscle, bepridil probably has two sites of action, one outside the cell (presumably on or associated with the slow Ca2+ channel) and a second site inside the cell. On the other hand, in vascular smooth muscle cells, bepridil may act only on an external site.