The distribution and single channel properties of acetylcholine (ACh) receptors in human myotubes grown in tissue culture have been examined. Radioautography of myotubes labelled with [125I]α-bungarotoxin showed that ACh receptors are distributed uniformly over the myotube surface at a density of 3.9 ± 0.5 receptors per square micrometre. Accumulations of ACh receptors (hot spots) were found rarely. The conductance and kinetics of ACh-activated channels were investigated with the patch-clamp technique. Cell-attached membrane patches were used in all experiments. A single channel conductance in the range 40-45 pS was calculated. No sublevels of conductance (substates) of the activated channel were observed. The distribution of channel open-times varied with ACh concentration. With 100 nM ACh, the distribution was best fitted by the sum of two exponentials, whereas with 1 μM ACh a single exponential could be fitted. The mean channel open-time at the myotube resting potential (ca. -70 mV, 22° C) was 8.2 ms. The distribution of channel closed-times was complex at all concentrations of ACh studied (100 nM to 10 μM). With densitizing doses of ACh (10 μM), channel openings occurred in obvious bursts; each burst usually appeared as part of a 'cluster' of bursts. Both burst duration and mean interval between bursts increased with membrane hyperpolarization. Individual channel open-times and burst durations showed similar voltage dependence (e-fold increase per 80 mV hyperpolarization), whereas both the channel closed-times within a burst and the number of openings per burst were independent of membrane potential.