1. Adenosine caused dose-dependent action potential abbreviation in multicellular guinea-pig atrial preparations, an action antagonized by glyburide (IC50, 31 microM) in both physiological and low-chloride superfusate. 2. When 5 mM ATP was included in pipettes for whole-cell voltage clamp of isolated guinea-pig atrial myocytes, adenosine (10 microM) increased the holding current at -40 mV from 41 +/- 8 to 246 +/- 31 pA (mean +/- S.E.M., P < 0.01), and glyburide (20 microM) returned the holding current to 69 +/- 11 pA (P < 0.01 vs. adenosine alone). Acetylcholine (10 microM) also increased the holding current, but its effects were not altered by glyburide. 3. Both adenosine and acetylcholine induced an additional current component in response to 500 ms voltage steps. Glyburide partially inhibited the adenosine-induced current, but did not alter the effect of acetylcholine. In the presence of maximally effective acetylcholine concentrations, adenosine increased membrane conductance (P < 0.01), although to a lesser extent than in the absence of acetylcholine. 4. Single K+ channel activity was seen in only one of eight cell-attached patches in the absence of adenosine or acetylcholine (0.5 mM Ba2+ in bath and pipette solutions). With acetylcholine (10 microM) in the pipette, inwardly rectifying channels (conductance, 41 +/- 5 pS) were seen in five of six patches. With adenosine (10 microM) in the pipette, single-channel activity was seen in twelve of fourteen patches with two populations of channels, one similar to that induced by acetylcholine and another higher-conductance channel (72 +/- 5 pS) that showed less inward rectification. Glyburide (20 microM) suppressed the high-conductance channel (68 +/- 2 pS) leaving a single channel type with a conductance of 36 +/- 5 pS and strong inward rectification. 5. We conclude that K+ATP channels contribute to the electrophysiological actions of adenosine on guinea-pig atrium in the presence of physiological intracellular ATP levels, and may therefore play a role in the cardiac electrophysiological effects of adenosine in the absence of myocardial ischaemia.