TRPM2 is a Ca(2+)-permeable non-selective cation channel that uniquely is activated by intracellular ADP-ribose. To date, only one pharmacological blocker of this channel, namely flufenamic acid (FFA), has been described. Here we demonstrate, using patch clamp electrophysiology, that the antifungal imidazoles clotrimazole and econazole inhibit ADP-ribose-activated currents in HEK-293 cells expressing recombinant human TRPM2 (hTRPM2). For both compounds, all concentrations in a range from 3 microM to 30 microM produced an essentially complete inhibition of the TRPM2-mediated current. The rate of current antagonism was dependent on the concentration applied, with higher concentrations producing faster block. In addition, decreasing extracellular pH accelerated inhibition of TRPM2 by both clotrimazole and econazole; extracellular alkalisation produced the converse effect. Additional experiments indicated hTRPM2 activation was required for the antagonism of either compound to develop, and that neither compound blocked from the intracellular face of the plasma membrane. ADP-ribose-activated whole-cell and single-channel currents in the rat insulinoma cell-line CRI-G1 were also antagonised by clotrimazole. Contrary to the observations made with hTRPM2, antagonism in CRI-G1 cells could be largely reversed following clotrimazole removal. These experiments suggest that imidazole antifungals may be useful tool antagonists for future studies of TRPM2 function.