Background and purpose: ATP‐sensitive potassium channels (KATPchannels) in beta cells are a major target for insulinotropic drugs. Here, we studied the effects of selected stimulatory and inhibitory pharmacological agents in islets lacking KATPchannels.Experimental approach: We compared insulin secretion (IS) and cytosolic calcium ([Ca2+]c) changes in islets isolated from control mice and mice lacking sulphonylurea receptor1 (SUR1), and thus KATPchannels in their beta cells (Sur1KO).Key results: While similarly increasing [Ca2+]cand IS in controls, agents binding to site A (tolbutamide) or site B (meglitinide) of SUR1 were ineffective inSur1KO islets. Of two non‐selective blockers of potassium channels, quinine was inactive, whereas tetraethylammonium was more active inSur1KO compared with control islets. Phentolamine, efaroxan and alinidine, three imidazolines binding to KIR6.2 (pore of KATPchannels), stimulated control islets, but only phentolamine retained weaker stimulatory effects on [Ca2+]cand IS inSur1KO islets. Neither KATPchannel opener (diazoxide, pinacidil) inhibitedSur1KO islets. Calcium channel blockers (nimodipine, verapamil) or diphenylhydantoin decreased [Ca2+]cand IS in both types of islets, verapamil and diphenylhydantoin being more efficient inSur1KO islets. Activation of α2‐adrenoceptors or dopamine receptors strongly inhibited IS while partially (clonidine > dopamine) lowering [Ca2+]c(control >Sur1KO islets).Conclusions and implications: Those drugs retaining effects on IS in islets lacking KATPchannels, also affected [Ca2+]c, indicating actions on other ionic channels. The greater effects of some inhibitors inSur1KO than in control islets might be relevant to medical treatment of congenital hyperinsulinism caused by inactivating mutations of KATPchannels.