Human TRPC5 (hTRPC5) is a putative cationic channel that was cloned from a region of X chromosome associated with mental retardation (Sossey-Alaoui et al, 1999). We produced hTRPC5 stably expressing HEK-293 cells under tetracycline-regulation. Over-expression of hTRPC5 was confirmed by real time RT-PCR and western blotting. Functional hTRPC5 were measured by calcium imaging and electrophysiology. In tetracycline-induced cells, no basal activity was evident but activity was induced by carbachol stimulation of muscarinic receptors independently of calcium release. This is “receptor-activation”, as described for mouse TRPC5 (Strubing et al, 2001). In addition, and in the absence of receptor stimulation, extracellular 0.1 mM gadolinium activated TRPC5, an effect that was mimicked by 5-10 mM extracellular calcium with intracellular calcium buffered. We refer to this as “external ionic-activation”. Using whole-cell recording, we demonstrated that TRPC5 was also activated by modest elevation of [Ca²⁺]_i in the absence of GTP – “calcium-activation”. A putative fourth activation mechanism is a signal from depleted intracellular calcium stores. Consistent with this idea, hTRPC5 was activated by a standard protocol in which calcium stores were first depleted by treatment of cells with thapsigargin in calcium free solution, followed by re-addition of 1.5 mM calcium to the bath solution. This result can be mimicked by replacing calcium with 0.5 mM barium, an effect that was difficult to explain by calcium activation. TRPC1 is thought to participate in a physiological heteromultimeric assembly with TRPC5 (Hofmann et al, 2002). When it was co-expressed with hTRPC5, the store-operated property of hTRPC5 was retained, although the amplitude of the signal was smaller. We also tested different activation signals on isolated cells, and thus on the single expression levels of these cells. Multiplicity of TRPC5 activation was evident: the TRPC5dependent “store-operated” calcium re-entry signal and “receptoractivation” both occurred in a single cell, as did “external-ionic” and “receptor” activation. In conclusion, hTRPC5 is activated by a range of stimuli, avoiding dependence on a single critical activator like TRPV4 (Vriens et al, 2004) and unlike many other ion channels.