1. In mouse pancreatic beta-cells the role of cytosolic nucleotides in the regulation of the sulphonylurea sensitivity of the adenosine 5'-triphosphate-sensitive K+ channel (KATP-channel) was examined. Patch-clamp experiments with excised inside-out membrane patches were carried out using an experimental protocol favouring phosphorylation of membrane proteins. 2. In the absence of Mg2+, the KATP-channel-inhibiting potency of cytosolic nucleotides decreased in the order ATP = adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S) > adenosine 5'-diphosphate (ADP) > adenosine 5'-O-(2-thiodiphosphate) (ADP beta S) = adenylyl-imidodiphosphate (AMP-PNP) > 2'-deoxyadenosine 5'-triphosphate (dATP) > uridine 5'-triphosphate (UTP) > 2'-deoxyadenosine 5'-diphosphate (dADP) > guanosine 5'-triphosphate (GTP) > guanosine 5'-diphosphate (GDP) > uridine 5'-diphosphate (UDP). 3. In the presence of Mg2+, the inhibitory potency of cytosolic nucleotides decreased in the order ATP gamma S > ATP > AMP-PNP > ADP beta S > dATP > UTP. In the presence of Mg2+, the KATP-channels were activated by dADP, GTP, GDP and UDP. 4. Tolbutamide inhibited the KATP-channels not only in the presence but also in the prolonged absence of Mg2+. In nucleotide-free solutions, the potency of tolbutamide was very low. When about half of the KATP-channel activity was inhibited by ATP, AMP-PNP, ADP beta S or ADP (absence of Mg2+), the potency of tolbutamide was increased. 5. Tolbutamide (100 microM) slightly enhanced the channel-inhibiting potency of AMP-PNP and inhibited the channel-activating effect of MgGDP in a non-competitive manner. 6. Channel activation by MgGDP (0.5 mM) competitively antagonized the inhibitory responses to AMP-PNP (1 MicroM- 1 mM). This effect of GDP was neutralized by tolbutamide (100 MicroM).7. The stimulatory effect of 0.5 mM MgGDP was neutralized by 200 MicroM AMP-PNP. Under these conditions the potency of tolbutamide was much higher than in the presence of 0.5 mM MgGDP alone or in the absence of any nucleotides.8. dADP (0.3-1 mM) increased the potency of tolbutamide. Additional application of 200 MicroM AMPPNP caused a further increase in the potency of tolbutamide.9. In conclusion, in the simultaneous presence of inhibitory and stimulatory nucleotides, binding of sulphonylureas to their receptor causes direct inhibition of channel activity, non-competitive inhibition of the action of stimulatory nucleotides and interruption of the competitive interaction between stimulatory and inhibitory nucleotides. The latter effect increases the proportion of KATP- channels staying in the nucleotide-blocked state. In addition, this state potentiates the direct effect of sulphonylureas.