The vomeronasal organ (VNO) is a chemosensory organ present in most mammals. The main function of the VNO is to detect pheromones regulating the physiology and social behavior of the organism. Chemosensory neurons in the VNO, named vomeronasal sensory neurons (VSNs), express vomeronasal receptors in the specialized knob/microvilli region where signal transduction takes place. Signal transduction cascade is poorly understood in VSNs and some steps still remain obscure. The two calcium-activated chloride channels, TMEM16A and TMEM16B, are expressed in the knob/microvilli region of VSNs [1,2,3], however, their physiological role in VSNs is still controversial. We have previously reported that TMEM16A is an essential component to mediate the calcium-activated chloride currents in VSNs [4]. Moreover, a recent report showed that deletion of both TMEM16A and TMEM16B proteins lead to a reduced capability of spontaneous and evoked firing in VSNs, but did not modify male-male aggression, a VNO-mediated behavior [5]. Taking advantage of Tmem16a conditional knockout (cKO) and Tmem16b KO mice, we aimed to evaluate the individual contribution of TMEM16A and TMEM16B proteins to the physiology of VSNs. By immunohistochemistry, we confirmed the expression of TMEM16B protein in Tmem16a cKO mice and the expression of TMEM16A protein in Tmem16b KO mice. Then, we evaluated calcium-activated chloride currents in VSNs using the whole-cell patch-clamp technique. We confirmed that TMEM16A is the principal contributor to calcium-activated chloride currents in VSNs, while TMEM16B contribution to those currents seems to be minor (WT-16AcKO: p<0.01; Dunn–Hollander–Wolfe after Krustal-Wallis). To check the role of calcium-activated chloride currents in VSNs, we first evaluated voltage-gated currents and passive membrane properties in WT, Tmem16a cKO and Tmem16b KO VSNs (n=75). We did not find differences between the three groups (p=0.21; Krustal-Wallis), indicating that TMEM16A and TMEM16B proteins do not contribute to passive membrane properties or to voltage-gated currents in VSNs. Finally, we investigated the spontaneous (n=136 cells) and evoked activity (n=54 cells) in VSNs from acute slices of VNO, using the loose-patch recording technique. We found that deletion of TMEM16A protein affects spontaneous activity pattern modulating the inter-spike interval (ISI) (p<0.001; Kolmogorov-Smirnov), while deletion of TMEM16B protein did not affect spontaneous activity. Furthermore, deletion of either Tmem16a or Tmem16b did not affect the frequency of spontaneous activity (p=0.62; Krustal-Wallis). Then, we evaluated the spiking properties of VSNs in response to urine stimulation. We found that VSNs from both KO mice conserved the capability of response to urine stimulation when compared with WT. We did not find an alteration in frequency of response or response duration (p=0.71; Krustal-Wallis), while we found differences in the ISI distribution (p<0.001; Kolmogorov-Smirnov), meaning that either deletion of TMEM16A or TMEM16B protein alters the spiking pattern of VSNs during the response to urine. We concluded that, while TMEM16A is the main contributor to calcium-activated chloride currents in VSNs, both TMEM16A and TMEM16B proteins modulate action potential firing during spontaneous and evoked activity. Also, our results indicate that TMEM16A and TMEM16B proteins can work as heterodimers in the knob/microvilli region of VSNs.