Arrestins bind active phosphorylated G protein-coupled receptors, terminating G protein activation. Receptor-bound non-visual arrestins interact with numerous partners, redirecting signaling to alternative pathways. Arrestins also have nuclear localization and nuclear exclusion signals and shuttle between the nucleus and the cytoplasm. Constitutively shuttling proteins often redistribute their interaction partners between the two compartments. Here we took advantage of the nucleoplasmic shuttling of free arrestins and used a "nuclear exclusion assay" to study their interactions with two proteins involved in "life-and-death" decisions in the cell, the kinase JNK3 and the ubiquitin ligase Mdm2. In human embryonic kidney 293 cells green fluorescent protein (GFP)-JNK3 and GFP-Mdm2 predominantly localize in the nucleus, whereas visual arrestin, arrestin2(Q394L) mutant equipped with the nuclear exclusion signal, and arrestin3 localize exclusively to the cytoplasm. Coexpression of arrestins moves both GFP-JNK3 and GFP-Mdm2 to the cytoplasm. Arrestin mutants "frozen" in the basal conformation are the most efficacious. Thus, arrestins in their basal state interact with JNK3 and Mdm2, suggesting that arrestins are likely "preloaded" with their interaction partners when they bind the receptor. Robust interaction of free arrestins with JNK3 and Mdm2 and their ability to regulate subcellular localization of these proteins may play an important role in the survival of photoreceptors and other neurons, as well as in retinal and neuronal degeneration.