PURPOSE: To characterize the retinal expression and localization of Kcne2, an ancillary (β) ion-channel subunit with an important role in fine-tuning cellular excitability. METHODS: We analyzed available single-cell transcriptome data from tens of thousands of murine retinal cells for cell-type-specific expression of Kcne2 using state-of-the-art bioinformatics techniques. This evidence at the transcriptome level was complemented with a comprehensive immunohistochemical characterization of mouse retina (C57BL/6, ages 8-12 weeks) employing co-labeling techniques and cell-type-specific antibody markers. We furthermore examined how conserved the Kcne2 localization pattern in the retina was across species by performing immunostaining on zebrafish, cowbird, sheep, mice, and macaque. RESULTS: Kcne2 is distinctly expressed in cone photoreceptors and rod bipolar cells. At a subcellular level, the bulk of Kcne2 immunoreactivity can be observed in the outer plexiform layer. Here, it localizes into cone pedicles and likely the postsynaptic membrane of the rod bipolar cells. Thus, the vast majority of Kcne2 immunoreactivity is observed in a thin band in the outer plexiform layer. In addition to this, faint Kcne2 immunoreactivity can also be observed in cone inner segments and the somata of a small subset of cone ON bipolar cells. Strikingly, the localization of Kcne2 in the outer plexiform layer was preserved among all of the species studied, spanning at least 300 million years of evolution of the vertebrate kingdom. CONCLUSIONS: The data we present here suggest an important and specific role for Kcne2 in the highly specialized photoreceptor-bipolar cell synapse.