Our previous studies using electrophysiological and microfluorimetry techniques have suggested that several Ca²⁺ influx pathways exist in rat retinal arterioles (Scholfield et al. 2007). These include voltage-dependent L-type Ca²⁺ channels activated by cell membrane depolarization and non-voltage-gated receptor- and store-operated Ca²⁺ channels. Recent work has identified members of the transient receptor potential (TRP) channel superfamily as important molecular entities underlying non-voltage-gated Ca²⁺ influx pathways in different tissues, including those of the vasculature (Inoue et al. 2006). In the present study we sought to identify systematically the TRP channel subtypes expressed in rat retinal arterioles. Male Sprague-Dawley rats (200-300g) were anaesthetized with CO₂ and killed by cervical dislocation. Retinas were removed, arterioles mechanically isolated and TRP channel mRNA expression evaluated by RT-PCR. Detection of TRP channel family members was verified in cDNA reverse-transcribed from three different RNA isolations (10 vessels per isolation). Rat brain and kidney samples were used as positive controls. Immunofluorescence staining with commercially available polyclonal antibodies was also used to test for cell-specific expression of TRP channels in retinal arterioles embedded within retinal flatmount preparations. To ensure that the images collected originated from the arterioles the retinas were counter-stained with propidium iodide nuclear stain. Using RT-PCR, 13 TRP channel subtypes were detected in retinal arterioles: TRPC1, TRPC3, TRPC4, TRPC7, TRPV1, TRPV2, TRPV4, TRPM1, TRPM2, TRPM3, TRPM7, TRPML1 and TRPML3. Lack of expression of other TRP channels could not be attributed to primer design since all of the other TRP channel family members could be detected in brain or kidney. Immunofluorescence labelling revealed a punctuate distribution of TRPV2 throughout the vascular smooth muscle cell layer of retinal arterioles, while TRPV4 was specifically localized to the plasma membrane of endothelial cells. Consistent with the RT-PCR results, TRPC5 could not be detected in retinal arterioles. This was not due to a lack of antibody reactivity because intense staining was observed in the ganglion cell layer of the retina. This study defines for the first time the main TRP channel subtypes expressed in retinal arterioles. These channels represent promising candidates for non-voltage-gated Ca²⁺ entry pathways within the retinal microcirculation.