High-voltage activated Ca channels participate in multiple cellular functions, including transmitter release, excitation, and gene transcription. Ca channels are heteromeric proteins consisting of a pore-forming α1 subunit and auxiliary α2δ and β subunits. Although there are reports of α2δ4 subunit mRNA in the mouse retina and localization of the α2δ4 subunit immunoreactivity to salamander photoreceptor terminals, there is a limited overall understanding of its expression and localization in the retina. α2δ4 subunit expression and distribution in the mouse and rat retina were evaluated by using reverse transcriptase polymerase chain reaction, western blot, and immunohistochemistry with specific primers and a well-characterized antibody to the α2δ4 subunit. α2δ4 subunit mRNA and protein are present in mouse and rat retina, brain, and liver homogenates. Immunostaining for the α2δ4 subunit is mainly localized to Müller cell processes and endfeet, photoreceptor terminals, and photoreceptor outer segments. This subunit is also expressed in a few displaced ganglion cells and bipolar cell dendrites. These findings suggest that the α2δ4 subunit participates in the modulation of L-type Ca2+ current regulating neurotransmitter release from photoreceptor terminals and Ca2+-dependent signaling pathways in bipolar and Müller cells. J. Comp. Neurol. 521:2486–2501, 2013. © 2013 Wiley Periodicals, Inc.