1. The effects of extracellular adenosine 5'-triphosphate (ATP) on the acetylcholine (ACh) channel opening activity was studied in dissociated rat skeletal muscle cells using patch-clamp recording techniques in cell-attached configuration. 2. With 10 microM-ATP in the recording pipette, the spontaneous opening frequency on the the alpha-bungarotoxin-sensitive ACh channel increased significantly from 0.3 to 4.7 s-1, although the opening frequency was not as high as that activated by very low (0.4 microM) ACh concentrations (64 s-1). 3. Spontaneous ACh channel openings, and ATP-associated and ACh-activated channel openings had similar single-channel conductances, 55, 59 and 56 pS, respectively. 4. ATP-associated events and spontaneous ACh channel opening events had similar mean channel open durations (0.6 ms); however, these values were considerably shorter than the duration of ACh-activated events (2 ms). 5. Pre-treatment with alpha-bungarotoxin (100 nM) blocked spontaneous ACh channel openings, ATP-associated openings and ACh-activated openings. 6. When delivered through a separate drug pipette after the formation of a gigaseal, ATP increased ACh-activated single-channel open probability in a dose-dependent fashion. 7. The increase in channel open probability was due primarily to the increase in channel opening frequency. ATP did not significantly alter the mean channel open duration or the single-channel conductance. 8. The ATP analogue adenosine-5'-O-(3-thiotriphosphate) (ATP-gamma-S) also enhanced ACh-activated channel open probability with relatively less potency. ADP, AMP and adenosine (up to 1 mM) did not significantly increase ACh channel open probability. 9. It is concluded that ATP in the micromolar range facilitates both spontaneous and agonist-activated ACh channel opening. The facilitation is due to ATP itself and not to products of ATP hydrolysis. The facilitatory actions of ATP on ACh channels are manifested by the increase in the channel opening frequency, and they may be mediated by an intracellular second messenger.