In terrestrial vertebrates, the vomeronasal organ (VNO) detects and transduces pheromone signals. VNO activation is thought to be mediated by the transient receptor potential C2 (TRPC2) channel. The aberrant behavioural phenotypes observed in TRPC2-/- mice are generally attributed to the lost VNO function. Recently, calcium-activated chloride channels have been shown to contribute to VNO activation. Here we show that CACCs can be activated in VNO slice preparations from the TRPC2-/- mice and this activation is blocked by pharmacological agents that inhibit intracellular Ca(2+) release. Urine-evoked Cl(-) current is sufficient to drive spiking changes in VNO neurons from both wild-type (WT) and TRPC2-/- mice. Moreover, blocking Cl(-) conductance essentially abolishes VNO activation in WT neurons. These results suggest a TRPC2-independent signalling pathway in the VNO and the requirement of calcium-activated chloride channels currents to mediate pheromone activation. Our data further suggest that TRPC2-/- mice retain partial VNO function.