Non-genomic effects of steroidal substances, occurring on sub- second time scales, are often mediated by cell surface receptors, including ion channels. We found that the steroidal substance pregnenolone sulfate reversibly activates the ion channel TRPM3a2 on a millisecond time scale. We also found that nifedipine is capable of activating TRPM3a2 channels. Interestingly, TRPM3a2 channels are inhibited by extracellular Na+ ions. Accordingly, Na+ ions are only poorly conducted by TRPM3a2 channels. To the contrary, divalent cations permeate TRPM3a2 channels well. Measurement of the fractional Ca2+ current indicated that approx. 24% of the total inward current through TRPM3a2 is carried by Ca2+ . We identified membrane channels activated by pregnenolone sulfate in the b-cell derived cell line INS1 and in mouse pancreatic b-cells. These endogenous channels are likely encoded by TRPM3, as they shared all biophysical and pharmacological characteristics of recombinant TRPM3a2. Indeed, we could detect TRPM3 specific transcripts in pancreatic islets and INS1 cells using RT-PCR and Northern blotting, respectively. Our data therefore indicate that TRPM3 encodes novel ionotropic steroid receptors in pancreatic b-cells.