The ATP-sensitive K+(KATP) channels are composed of the pore-forming K+channel Kir6.0 and different sulfonylurea receptors (SURs). SUR1, SUR2A, and SUR2B are sulfonylurea receptors that are characteristic for pancreatic, cardiac, and vascular smooth muscle–type KATPchannels, respectively. The structural elements of SURs that are responsible for their different characteristics have not been entirely determined. Here we report that the 42 amino acid segment at the C-terminal tail of SURs plays a critical role in the differential activation of different SUR-KATPchannels by ADP and diazoxide. In inside-out patches of human embryonic kidney 293T cells coexpressing distinct SURs and Kir6.2, much higher concentrations of ADP were needed to activate channels that contained SUR2A than SUR1 or SUR2B. In all types of KATPchannels, diazoxide increased potency but not efficacy of ADP to evoke channel activation. Replacement of the C-terminal segment of SUR1 with that of SUR2A inhibited ADP-mediated channel activation and reduced diazoxide modulation. Point mutations of the second nucleotide-binding domains (NBD2) of SUR1 and SUR2B, which would prevent ADP binding or ATP hydrolysis, showed similar effects. It is therefore suggested that the C-terminal segment of SUR2A possesses an inhibitory effect on NBD2-mediated ADP-induced channel activation, which underlies the differential effects of ADP and diazoxide on KATPchannels containing different SURs.