TRPM3 is a poorly understood member of the TRP channel family. The splice variant TRPM3a2, recombinantly expressed in HEK293 cells, is activated by the steroidal substance pregnenolone sulphate. Since TRPM3a2 channels are highly permeable for Ca2+ , stimulation of cells expressing TRPM3a2 with pregnenolone sulfate leads to a rise of the intracellular Ca2+ concentration. Exploiting this fact we screened cell lines for pregnenolone sulfate induced Ca2+ signals and found such signals in GH3 and INS1 cells. Both cell lines are derived from rat endocrine organs, GH3 cells originate from the anterior pituitary and INS1 cells from pancreatic b-cells. Accordingly, we found pregnenolone sulfate induced Ca2+ signals in mouse pituitary cells and pancreatic islets cells in primary culture. Co- localization of steroid-induced Ca2+ signals with immunostaining for hormones demonstrated that pancreatic b-cells, but not a- cells, react to pregnenolone sulfate. Only approximately 20% of the cells from mouse pituitary glands reacted to the steroid. In all cell types studied, the endogenous steroid-activated channels displayed the key biophysical and pharmacological properties of recombinant TRPM3a2, indicating that they are encoded by TRPM3.