Rod tetrameric arrestin 1 (tet-ARR1), stored in the outer nuclear layer/inner segments in the dark, modulates photoreceptor synaptic activity; light exposure stimulates a reduction via translocation to the outer segments for terminating G-protein coupled phototransduction signaling. Here, we test the hypothesis that intraretinal spin-lattice relaxation rate in the rotating frame (1/T1r), an endogenous MRI contrast mechanism, has high potential for evaluating rod tet-ARR1 and its reduction via translocation. Dark-and light-exposed mice (null for the ARR1 gene, overexpressing ARR1, diabetic, or wild type with or without treatment with Mn 2+ , a calcium channel probe) were studied using 1/T1r MRI. Immunohistochemistry and single-cell recordings of the retinas were also performed. In wild-type mice with or without treatment with Mn 2+ , 1/T1r of avascular outer retina (64% to 72% depth) was significantly (P < 0.05) greater in the dark than in the light; a significant (P < 0.05) but opposite pattern was noted in the inner retina (<50% depth). Light-evoked outer retina D1/T1r was absent in ARR1-null mice and supernormal in overexpressing mice. In diabetic mice, the outer retinal D1/T1r pattern suggested normal darkto-light tet-ARR1 translocation and chromophore content, conclusions confirmed ex vivo. Light-stimulated D1/ T1r in inner retina was linked to changes in blood volume. Our data support 1/T1r MRI for noninvasively assessing rod tet-ARR1 and its reduction via protein translocation, which can be combined with other metrics of retinal function in vivo.-Berkowitz, B. A., Gorgis, J., Patel, A., Baameur, F., Gurevich, V. V., Craft, C. M., Kefalov, V. J., Roberts, R. Development of an MRI biomarker sensitive to tetrameric visual arrestin 1 and its reduction via light-evoked translocation in vivo. FASEB J. 29, 554-564 (2015). www.fasebj.org