The TRPM3 gene encodes for poorly characterized cation selective ion channels with unknown functions. Two factors contribute to the difficulties encountered in assigning a function to these channels: The lack of selective pharmacological tools and the absence of demonstrated currents through these channels in native tissues and cells. Using fluorometric Ca²⁺ measurements and electrophysiology, we show that steroidal compounds selectively activate channels encoded by the TRPM3a2 splice variant. The highly similar splice variant TRPM3a1 that differs only in 13 amino acids of the primary sequence was inhibited by these substances. The closely related channel TRPM7 was not affected at all. Steroidal compounds activated TRPM3a2 encoded channels quickly (less than 3 s) and reversibly. They are only effective when applied extracellularly. These data show that no genomic effect and probably no intracellular transduction cascade is involved in the activation of TRPM3a2 channels by steroids. We demonstrate that steroidal compounds can be used as a pharmacological tool to identify endogenously expressed channels with TRPM3a2-like properties in cell lines and in native murine cells maintained in primary culture.