Transient receptor potential vanilloid (TRPV) channels are members of a superfamily of non-selective cation channels possessing a vast range of physiological functions. Our aim was to fully characterise the molecular and functional expression of TRPV3 and TRPV4 channels in the retinal microvascular endothelium. Retinal microvascular endothelial cells (RMECs) were cultured from bovine retina. mRNA transcripts were detected for TRPV3 and V4 using RT-PCR. Protein expression was confirmed by Western blot analysis. Fura-2-based Ca2+ microfluorimetry was used to test for the functional expression of TRPV3 and V4 in RMECs using a selection of appropriate agonists and antagonists. The TRPV3/TRPA1 activator, carvacrol (100uM), evoked consistent [Ca2+]i responses in RMECs (0.2±0.03 R340/380, mean±SEM, n=5), which were reduced, but not abolished in the presence of the TRPA1 inhibitor, HC-030031 (10µM) (0.14±0.01 R340/380, n=6; p<0.05 v carvacrol alone). The functional expression of TRPV4 channels was tested using the selective activator GSKA (100nM). This drug elicited a transient rise in [Ca2+]i (1.88±0.62 R340/380, n=13) that was blocked by pre-incubation with the specific TRPV4 antagonist, HC067047(1uM) (0.06±0.04 R340/380, n=6; p<0.01 v. GSKA alone). The cellular localisation of TRPV3 and TRPV4 channels was subsequently examined in bovine retinal wholemounts and isolated RMECs using immunohistochemistry. TRPV3 was located predominantly in the nuclei of endothelial cells in retinal wholemounts with some punctate staining in the cytoplasm. No differences in localisation were observed in endothelial cells of arteries, capillaries and veins. RMECs also showed predominantly nuclear staining for TRPV3. TRPV4 was found to be located in both the nuclei and cytoplasm of endothelial cells lining the vasculature in retinal wholemounts. Interestingly sparse, punctate TRPV4 staining was noted in endothelial cells on the arterial side of the circulation, while those on the venous side presented with noticeably increased TRPV4 staining in both the nuclei and cytoplasm. In isolated RMECs, TRPV4 was localised to both the nucleus as well as tubular-like structures in the cytoplasm. Localisation patterns for each channel were confirmed using multiple antibodies to different epitopes and the specificity of staining was confirmed using blocking antigens. This study provides molecular and functional evidence for the expression of TRPV3 and V4 in bovine retinal endothelial cells. Further research is now required to elucidate their physiological significance.