Recent studies in platelets and T-cells have reported the involvement of phosphoinositide-3-kinase (PI-3-K) in calcium entry that is independent of regulation by intracellular stores. A cell permeable analogue of PIP3 namely dioctanoyl-PIP3 (DiC8-PIP3) induces cation entry in both T-cells and platelets. DiC8-PIP3 induces platelet aggregation though the full mechanisms are not established. Platelets express the transient receptor potential canonical (TRPC)1 and TRPC6 cation channels and we have suggested TRPC6 to be the major non-store regulated channel. TRPC6 also contains a p85 PI-3-K recognition motif. In this study we investigated a possible link between DiC8-PIP3 and TRPC6. Platelet aggregation and Ca2+ elevation were carried out using standard techniques. Akt activation was estimated using Western blotting. Transient transfection of desired proteins was carried out in QBI-293 cells using 30-60 ug of plasmid DNA to 70% confluent cells using the standard calcium phosphate technique. After 24 hrs cells were provided with fresh medium and experiments carried out 48 hrs later after labelling of cells with Fura2. Cytosolic calcium levels were monitored using 340/380 nm ratio fluorescence measurements. DiC8-PIP3 induces platelet aggregation with similar potency in either Ca2+ or Ba2+ containing medium and induces Ca2+ or Ba2+ entry without release from intracellular stores. DiC8-PIP3 also induced the activation of Akt in platelets as determined using an anti-phospho-Akt antibody suggesting that it activates other PIP3 effectors. Studies on hTRPC6 was carried out in QBI-293 cells. Fura2 loaded QBI-293 cells show typical responses of Ca2+ release and Ca2+ entry in response to carbachol addition and there was no response of Ca2+ elevation when the diacylglycerol analogue OAG was added. Over-expression of hTRPC6 resulted in an increase of OAG induced entry of Ca2+ / Ba2+ signifying the properties of over-expressed hTRPC6. Western blotting using anti-TRPC6 antibodies confirmed the over-expression. Stimulation of hTRPC6 cells with DiC8-PIP3 (20 uM) did not induce Ca2+ elevation suggesting the absence of a direct effect on hTRPC6. A recent study has suggested that TRPC6 can be tyrosine phosphorylated leading to positive modulation of channel activity. However co-expression of either hSYK or hFYN did not result in any stimulation of Ca2+ elevation with DiC8-PIP3 whilst the response to OAG was maintained. These results suggest that the non-store regulated, DiC8-PIP3 stimulation of cation entry in platelets most likely results from an indirect stimulation of cation entry channels via other PIP3 effectors.