Bupivacaine and its quaternary derivative, bupivacaine methiodide, were studied on acetylcholine (ACh)-activated single-channel currents recorded in myoballs from neonatal rat muscles using the patch-clamp technique. Under control conditions, the ACh-induced channels had three conductance states, 10, 20, and 33 pS, at a temperature of 10 degrees. The intermediate conductance state (20 pS) was the most prevalent. Moreover, an excessive number of very short events was observed which contributed to a deviation of the channel open-time distribution from a single-exponential function. At 20 degrees, the amplitude of these currents was increased (Q10 = 1.4), and the mean channel open time was decreased (Q10 = 3). Bupivacaine and its quaternary derivative (5-50 microM), when inside the patch micropipette with ACh, caused shortening of the channel open time, but the single-channel conductance remained unchanged at all concentrations studied. In the presence of bupivacaine, there was a loss of voltage dependence of the mean channel open time seen under control conditions; i.e., the shortening of the channel open time was more pronounced at more negative potentials. The plot of the reciprocal of mean channel open time versus bupivacaine concentration was linear. Similar effects were observed when bupivacaine was added to the bathing medium in both cell-attached and inside-out patch conditions, but in this case the onset of the drug action occurred at a later time and its potency was lower. Application of bupivacaine methiodide via the bathing medium after the establishment of the gigaohm seals, however, had no effect on the kinetics of ACh-activated single channels under both patch conditions (cell-attached and inside-out). The patch-clamp results indicated that the charged form of bupivacaine blocks the open state of ACh-activated ionic channels interacting with sites at the extracellular segment of the ACh receptor-ionic channel complex and creating a species with little or no conductance. A sequential model can be used to explain the interactions of these noncompetitive antagonists of the ACh receptor-ionic channel complex with the open channel. This interpretation of the action of bupivacaine and its quaternary analogue as open channel blockers also was reached based on an analysis of macroscopic events in nicotinic synapses of frog muscle.