Rationale: Canonical transient receptor potential (TRPC)-encoded non-selective cation channels (NSCCs) are crucial for many cellular responses in a variety of cells; however, their molecular expression and functional roles in airway smooth muscle cells (ASMCs) remain obscure. Objectives: To determine whether and TRPC1 and TRPC3 molecules could be important molecular constituents of native NSCCs controlling the resting membrane potential (Vm) and [Ca2+]i in freshly isolated normal and ovalbumin-sensitized/challenged mouse ASMCs. Methods: Western blotting, TR-PCR, single-channel recording, whole-cell current-clamp recording, and a fluorescence imaging system were used to determine TRPC expression, NSCC activity, resting Vm and resting [Ca2+]i. Specific individual TRPC antibodies and siRNAs were applied to test their functional roles. Results: TRPC1 and TRPC3 proteins and mRNAs were expressed in freshly isolated ASMC tissues. TRPC3 antibodies blocked the activity of NSCCs and hyperpolarized the resting Vm in ASMCs, while TRPC1 antibodies had no effect. TRPC3, but not TRPC1 gene silencing largely diminished NSCC activity, hyperpolarized the resting Vm, lowered the resting [Ca2+]i, and inhibited methacholine-induced increase in [Ca2+]i. In ovalbumin-sensitized/challenged ASMCs, NSCC activity was greatly augmented, resting Vm depolarized, and TRPC3 protein expression increased. Both TRPC1 and TRPC3 antibodies blocked the increased activity of NSCCs and membrane depolarization in ovalbumin-sensitized/challenged cells. Conclusions: TRPC3 is an important molecular component of native NSCCs contributing to the resting Vm and [Ca2+]i in normal ASMCs, as well as membrane depolarization and hyperresponsiveness in ovalbumin-sensitized/challenged cells, whereas TRPC1-encoded NSCCs are only activated in ovalbumin-sensitized/challenged airway myocytes.