Located at the anterior portion of the nose, the paired vomeronasal organs (VNO) detect odors and pheromones. In vomeronasal sensory neurons (VSNs) odor responses are mainly mediated by phospholipase C (PLC), stimulation of which elevates diacylglycerol (DAG). DAG activates a transient receptor potential channel (TRPC2) leading to cell depolarization. In this study, we used a natural stimulus, urine, to elicit odor responses in VSNs and found urine responses persisted in TRPC2(-/-) mice, suggesting the existence of a TRPC2-independent signal transduction pathway. Using perforated patch-clamp recordings on isolated VSNs from wild-type (WT) and TRPC2(-/-) mice, we found a PLC inhibitor blocked urine responses from all VSNs. Furthermore, urine responses were reduced by blocking DAG lipase, an enzyme that produces arachidonic acid (AA), in WT mice and abolished in TRPC2(-/-) mice. Consistently, direct stimulation with AA activated an inward current that was independent of TRPC2 channels but required bath Ca(2+) and was blocked by Cd(2+). With the use of inside-out patches from TRPC2(-/-) VSNs, we show that AA activated a channel that also required Ca(2+). Together, these data from WT and TRPC2(-/-) mice suggest that both DAG and its metabolite, AA, mediate excitatory odor responses in VSNs, by activating two types of channels, a TRPC2 and a separate Ca(2+)-permeable channel.