Our previous studies have demonstrated that neither receptor endocytosis nor arrestin is required for ERK activation by the α2- adrenergic receptor (Wang, Q., Zhao, J., Brady, A. E., Feng, J., Allen, P. B., Lefkowitz, R. J., Greengard, P., and Limbird, L. E. (2004) Science 304, 1940-1944). The present studies address whether arrestin plays a role in determining the route of α2AR-evoked ERK signaling activation, taking advantage of endogenous expression of the α2AAR subtype in mouse embryonic fibroblasts (MEFs) and the availability of MEFs without arrestin expression (derived from Arr2,3-/- mice). Our data demonstrate that the endogenous α2AAR evokes ERK phosphorylation through both a Src-dependent and a Src-independent pathway, both of which are G protein dependent and converge on the Ras-Raf-MEK pathway. Arrestin is essential to recruit Src to this process, as α2AAR- mediated ERK signaling in Arr2,3-/- MEFs does not involve Src. Stimulation of α2AAR enhances arrestin-Src interaction and promotes activation of Src. α2 agonists have similar potencies in stimulating Src-dependent and Src-independent ERK phosphorylation in wild-type and Arr2,3-/- cells, respectively. However, Src-independent α2AAR-mediated ERK stimulation has both a longer duration of activation and a more rapid translocation of pERK into the nucleus when compared with Src-dependent activation. These data not only affirm the role of arrestin as an escort for signaling molecules such as Src family kinases but also demonstrate the impact of arrestin-dependent modulation on both the temporal and spatial properties of ERK activation. © 2006 by The American Society for Biochemistry and Molecular Biology, Inc.