TRPM3 and TRPM1 are closely related proteins of the large and diverse TRP cation channel family. Recently, we have established that TRPM3 is a divalent permeable steroid receptor that can be activated by extracellular application of the endogenously produced steroid pregnenolone sulfate (Wagner et al., 2008, Nature Cell Biol. 10, 1421-1430). However, little is known about the function and the properties of TRPM1 channels as it has not been shown conclusively that these proteins can form ion-conducting pores. Using overexpression of recombinant proteins in HEK293 cells and subsequent whole-cell patch clamping, we show here that overexpression of specific splice variants of TRPM1 proteins results in significant, but very small steroid-induced currents in transfected cells. In order to study the biophysical properties of TRPM1 channels, we employed two strategies: (1) Transplantation of the pore region of TRPM1 channels into TRPM3 channels and (2) deletion of a part of the cytosolic amino-terminus. Both strategies resulted in proteins that generated large, well resolved currents after steroid application in the overexpression system. We were thus able to show that the TRPM1 pore is permeable to Ca²⁺ ions, but – in sharp contrast to TRPM3 channels – does not conduct Zn²⁺ ions. Indeed, constructs containing the TRPM1 pore are inhibited by Zn²⁺. Furthermore, heteromultimeric channels made of wild-type TRPM3 and TRPM1 proteins are also inhibited by extracellular Zn²⁺ ions, in contrast to homomultimeric TRPM3 channels. Using Zn²⁺ as a pharmacological probe, we show that endogenously expressed steroid-activated channels in Ins-1 cells (a rat pancreatic ß-cell line) are partially sensitive to Zn²⁺, while the steroid-sensitive channels in mouse pancreatic ß-cells in primary culture are not, indicating that the steroid-sensitive channels may differ molecularly between Ins-1 and mouse pancreatic ß-cells. Interestingly, TRPM1 expression can readily be detected in Ins-1 cells.