TRPC6 is thought to be a Ca2+-permeable cation channel activated following stimulation of G-protein-coupled membrane receptors linked to phospholipase C (PLC). TRPC6 current is also activated by exogenous application of 1-oleoyl-acetyl-sn-glycerol (OAG) or by inhibiting 1,2-diacylglycerol (DAG) lipase activity using RHC80267. In the present study, both OAG and RHC80267 increased whole-cell TRPC6 current in cells from a human embryonic kidney cell line (HEK 293) stably expressing TRPC6, but neither compound increased cytosolic free Ca2+ concentration ([Ca2+]i) when the cells were bathed in high-K+ buffer to hold the membrane potential near 0 mV. These results suggested that TRPC6 channels have limited Ca2+ permeability relative to monovalent cation permeability and/or that Ca2+ influx via TRPC6 is greatly attenuated by depolarization. To evaluate Ca2+ permeability, TRPC6 currents were examined in extracellular buffer in which Ca2+ was varied from 0.02 to 20 mm. The results were consistent with a pore-permeation model in which Ca2+ acts primarily as a blocking ion and contributes only a small percentage (∼4%) to whole-cell currents in the presence of extracellular Na+. Measurement of single-cell fura-2 fluorescence during perforated-patch recording of TRPC6 currents showed that OAG increased [Ca2+]i 50–100 nm when the membrane potential was clamped at between −50 and −80 mV, but had little or no effect if the membrane potential was left uncontrolled. These results suggest that in cells exhibiting a high input resistance, the primary effect of activating TRPC6 will be membrane depolarization. However, in cells able to maintain a hyperpolarized potential (e.g. cells with a large inwardly rectifying or Ca2+-activated K+ current), activation of TRPC6 will lead to a sustained increase in [Ca2+]i. Thus, the contribution of TRPC6 current to both the kinetics and magnitude of the Ca2+ response will be cell specific and dependent upon the complement of other channel types.