1. The effects of mexiletine were evaluated on the ATP-sensitive K+ channel (K(ATP)) of rat skeletal muscle fibres using patch clamp techniques. The effects of mexiletine were studied on macropatch currents 20 s (maximally activated), 8 min (early stage of rundown) and 15 min (late stage of rundown) after excision in the absence or in the presence of internal ADP (50-100 microM) or UDP (500 microM). In addition, the effects of mexiletine were tested on single channel. 2. In the absence of ADP and UDP, mexiletine inhibited the current through maximally activated channels with an IC50 of -5.58+/-0.3 M. Nucleoside diphosphates shifted the current versus mexiletine concentration relationship to the right on the log concentration axis. UDP (500 microM) was more efficacious than ADP (50-100 microM) in this effect. 3. At the early stage of rundown, the sensitivity of the channel to mexiletine was reduced and nucleoside diphosphates, particularly UDP, antagonized the effect of mexiletine. At the late stage of rundown, mexiletine did not affect the currents. 4. At the single channel level, 1 microM mexiletine reduced the mean burst duration by 63% and prolonged the arithmetic mean closed time intervals between the bursts of openings without altering the open time and closed time distributions. Mexiletine did not affect the single channel conductance. 5. These results show that in skeletal muscle, mexiletine is a state-dependent K(ATP) channel inhibitor which either acts through the nucleotide binding site or a site allosterically coupled to it.