Spatiotemporal aspects of ERK activation are stimulus-specific and dictate cellular consequences. They are dependent upon dual-specificity phosphatases (DUSPs) that bind ERK via docking-domains and can both inactivate and anchor ERK in cellular compartments. Using high-throughput fluorescent microscopy in combination with a system where endogenous ERKs are removed and replaced with wild-type or mutated ERK2-GFP, we show that ERK2 activation responses to EGF and PKC are transient and sustained, respectively. PKC-mediated ERK2 activation is associated with prolonged nuclear localization in the dephosphorylated form, whereas EGF-stimulated ERK2 activation mediates only transient nuclear accumulation. Using siRNAs to nuclear-inducible DUSPs 1, 2 or 4 (alone or in combination), we demonstrate that all three of these enzymes contribute to the dephosphorylation of PKC (but not EGF)-activated ERK2 in the nucleus, but that they have opposing effects on localization. DUSP2 and 4 inactivate and anchor ERK2, while DUSP1 dephosphorylates ERK in the nucleus but allows its traffic back to the cytoplasm. Overexpression of either DUSP1, 2 or 4 prevented ERK2 activation, but only DUSP2 and 4 caused ERK2-GFP nuclear accumulation, or could be immunoprecipitated with ERK2. Furthermore, protein synthesis inhibition or replacement of wild type ERK2-GFP with docking domain mutants selectively increased PKC effects on ERK activity, and altered ERK2-GFP localization. These mutations also impaired the ability of ERK2-GFP to bind DUSP2 and 4. Together, our data reveal a novel, stimulus-specific and phosphatase-specific mechanism of ERK2 regulation in the nucleus by DUSP1, 2 and 4.