ATP-sensitive potassium (K-ATP) channels, composed of sulfonylurea receptor (SURx) and Kir6.x, play important roles by linking cellular metabolic state to membrane potential in various tissues. Pancreatic, cardiac, and vascular smooth muscle K-ATP channels, which consist of different subtypes of SURx, differ in their responses to cellular metabolic state, To explore the possibility that different interactions of SURx with nucleotides cause differential regulation of K-ATP, channels, we analyzed the properties of nucleotide-binding folds (NBFs) of SUR1, SUR2A, and SUR2B. SURx in crude membrane fractions was incubated with 8-azido[alpha-P-32]ATP Or 8-azido-[gamma-P-32]ATP under various conditions and was photoaffinity-labeled. Then, SURx was digested mildly with trypsin, and partial tryptic fragments were immunoprecipitated with antibodies against NBF1 and NBF2. Some nucleotide-binding properties were different among SUR subtypes as follows. 1) Mg2+ dependence of nucleotide binding of NBF2 of SUR1 was high, whereas those of SUR2A and SUR2B were low. 2) The affinities of NBF1 of SUR1 for ATP and ADP, especially for ATP, were significantly higher than those of SUR2A and SUR2B. 3) The affinities of NBF2 of SUR2B for ATP and ADP were significantly higher than those of SUR2A This is the first biochemical study to analyze and compare the nucleotide-binding properties of NBFs of three SUR subtypes, and our results suggest that their different properties may explain, in part, the differential regulation of KATP channel subtypes. The high nucleotide-binding affinities of SUR1 may explain the high ability of SUR1 to stimulate pancreatic K-ATP channels. It is also suggested that the C-terminal 42 amino acids affect the physiological roles of SUR2A and SUR2B by changing the nucleotide-binding properties of their NBFs.