Single-channel recording techniques have been used to examine interactions between anticholinesterases and ion channels activated by acetylcholine. Single-channel currents activated by 200 nM acetylcholine were recorded from cell-attached patches of BC3Hl mouse tumor cells grown in culture. Channels were recorded in the absence and presence of edrophonium (1–20 μM), neostigmine (2–20 μM), or pyridostigmine (10–200 μM). All three drugs shortened channel open time but did not alter single-channel current amplitude. Effects on channel open time were not secondary to inhibition of cholinesterase but appeared to involve direct interactions between anticholinesterase drugs and acetylcholine-activated channels. Drug concentrations calculated to reduce the time constant of open time distributions by 50% were 3.8 μM edrophonium, 4.6 μM neostigmine, or 97 μM pyridostigmine. Channel open time was decreased by edrophonium at concentrations comparable to those occurring during reversal of neuromuscular block, but it was reduced by neostigmine and pyridostigmine only at levels higher than those encountered clinically. Differences in interactions between anticholinesterases and acetylcholine-activated channels at the end plate may possibly account for some of the clinical differences between these drugs.