The putative ion channel subunits TRPC3, TRPC6 and TRPC7 comprise a structurally related subgroup of the family of mammalian TRPC channels. As is the case for the founding member of the TRPC family, Drosophila TRP, the ion channels formed by these proteins appear to be activated in some manner downstream of phospholipase C (PLC). Earlier studies indicating that TRPC3 could be activated by depletion of intracellular stores (i.e. that it is a store-operated channel, SOC) were subsequently shown to be attributable to constitutive activity of the channels. Studies on the mechanism of activation of TRPC6 and TRPC7 indicated that PLC-dependent activation involved diacylglycerol and was independent of G proteins or inositol 1,4,5-trisphosphate (IP3). Although TRPC3 can also be activated by diacylglycerols, there is evidence suggesting that these channels can be activated by IP3 and the IP3 receptor through a conformational coupling mechanism. We have re-examined the activation mechanism for TRPC3 in mammalian cells by using HEK293 cell lines stably expressing human TRPC3. Our data indicate that, like TRPC6 and TRPC7, TRPC3 is activated by PLC-generated diacylglycerol and is independent of G proteins or IP3. However, in an avian pre-B cell line, TRPC3 can function either as a diacylglycerol-activated channel, or as a SOC. The mechanism of regulation of TRPC3 in this cell line appears to be related to the level of expression of the protein.