Patch-clamp techniques were used to study the effects of internal nucleotide diphosphates on the KATP channel in mouse skeletal muscle. In inside-out patches, application of GDP (100 microM) and ADP (100 microM) reversibly increased the channel activity. In the presence of internal Mg2+ (1 mM), low concentrations of ADP (< 300 microM) enhanced channel activity and high concentrations of ADP (> 300 microM) limited channel opening while GDP activated the channel at all concentrations tested. In the absence of internal Mg2+, ADP decreased channel activity at all concentrations tested while GDP had no noticeable effect at submillimolar concentrations and inhibited channel activity at millimolar concentrations. GDP [beta S] (100 microM), which behaved as a weak GDP agonist in the presence of Mg2+, stimulated ADP-evoked activation whereas it inhibited GDP-evoked activation. The K+ channel opener pinacidil was found to activate the KATP channel but only in the presence of internal GDP, ADP and GDP [beta S]. The results are discussed in terms of the existence of multiple nucleotide binding sites, in charge of the regulation of the KATP channel.