In this study, we provide novel insight into the mechanism of how ERK2 can be sorted to different intracellular compartments and thereby mediate different responses. MEK1-activated ERK2 accumulated in the nucleus and induced proliferation. Conversely, MEK2-activated ERK2 was retained in the cytoplasm and allowed survival. Localization was a determinant for ERK2 functions since MEK1 switched from providing proliferation to be a mediator of survival when ERK2 was routed to the cytoplasm by the attachment of a nuclear export site. MEK1-mediated ERK2 nuclear translocation and proliferation were shown to depend on phosphorylation of S298 and T292 sites in the MEK1 proline-rich domain. These sites are phosphorylated on cellular adhesion in MEK1 but not MEK2. Whereas p21-activated kinase phosphorylates S298 and thus enhances the MEK1-ERK2 association, ERK2 phosphorylates T292, leading to release of active ERK2 from MEK1. On the basis of these results, we propose that the requirement of adhesion for cells to proliferate in response to growth factors, in part, may be explained by the MEK1 S298/T292 control of ERK2 nuclear translocation. In addition, we suggest that ERK2 intracellular localization determines whether growth factors mediate proliferation or survival and that the sorting occurs in an adhesion-dependent manner.—Skarpen, E., Flinder, L. I., Rosseland, C. M., rstavik, S., Wierød, L., Pedersen Oksvold, M., Skålhegg, B. S., Huitfeldt, H. S. MEK1 and MEK2 regulate distinct functions by sorting ERK2 to different intracellular compartments.