Clotrimazole (CLT) is an antifungal compound commonly used in over-the-counter medications for the topical treatment of fungal infections of the skin, vagina, and mouth. CLT exerts its antifungal actions by inhibiting P450-dependent enzymes. TRPM3a2 (1), a splice variant of TRPM3, is rapidly and reversibly activated by pregnenolone sulfate (PS) and nifedipine (1). Here, we demonstrate that CLT strongly potentiates the response of TRPM3a2 to pregnenolone sulfate (PS) stimulation. Direct application of CLT to TRPM3a2 has no effect, however preapplication of CLT followed by PS stimulation strongly potentiates the TRPM3a2 response. The potentiation by CLT is reversible, repetitive and independent of external calcium. At CLT concentrations above 1 mM, the intensity of potentiation does not dependent CLT dose but rather on PS concentration. The response to PS after priming with CLT was relatively slow, suggesting a modulation in the signaling cascade rather than a direct effect. We conclude that CLT potentiates the TRPM3a2 response in an indirect manner, possibly by preventing further metabolization of pregnenolone sulfate by inhibiting P450-dependent enzymes.(1) kindly provided by J. Oberwinkler, S. Phillip, V. Flockerzi, Homburg (Saar), Germany(2) Wagner TF, Loch S, Lambert S, Straub I, Mannebach S, Mathar I, Dufer M, Lis A, Flockerzi V, Philipp SE, Oberwinkler J, Transient receptor potential M3 channels are ionotropic steroid receptors in pancreatic beta cells, Nat Cell Biol. 2008;10(12):1383-4