In this study, we report the molecular cloning of cDNAs encoding three distinct isoforms of rat (r) TRP6 Ca2+ channels. The longest isoform, rTRP6A, contains 930 amino acid residues; rTRP6B lacks 54 amino acids (3-56) at the N terminus, and rTRP6C is missing an additional 68 amino acids near the C terminus. Transient transfection of COS cells with expression vectors encoding rTRP6A or rTRP6B increased Ca2+ influx and gave rise to a novel Ba2+ influx after activation of M5 muscarinic acetylcholine receptors. By contrast, passive depletion of intracellular Ca 2+ stores with thapsigargin did not induce Ba2+ influx in cells expressing rTRP6 isoforms. Ba2+ influx was also stimulated in rTRP6A-expressing cells after exposure to the diacylglycerol analog, 1-oleoyl-2-acetyl-sn-glycerol (OAG), but rTRP6B-expressing cells failed to show OAG-induced Ba2+ influx. Expression of a rTRP6 N-terminal fragment of rTRP6B or rTRP6A antisense RNA blocked M5 muscarinic acetylcholine receptor-dependent Ba2+ influx in COS cells that were transfected with rTRP6 cDNAs. Together these results suggest that rTRP6 participates in the formation of Ca2+ channels that are regulated by a G-protein-coupled receptor, but not by intracellular Ca2+ stores. In contrast to the results we obtained with rTRP6A and rTRP6B, cells expressing rTRP6C showed no increased Ca2+ or Ba2+ influxes after stimulation with carbachol and also did not show OAG-induced Ba2+ influx. Glycosylation analysis indicated that rTRP6A and rTRP6B are glycosylated in COS cells, but that rTRP6C is mostly not glycosylated. Together these results suggest that the N terminus (3-56 amino acids) is crucial for the activation of rTRP6A by diacylglycerol and that the 735-802 amino acid segment located just downstream from the 6th transmembrane segment may be required for processing of the rTRP6 protein.