The dephosphorylation of the MAP kinase (MAPK) Thr-X-Tyr activation loop motif is the most direct molecular mechanism that negatively regulates MAPK catalytic activity. Maximal activity of MAPKs is achieved by keeping their activation loop-Thr and -Tyr residues phosphorylated, although some kinase activity is preserved after the partial dephosphorylation of the motif by Ser/Thr-phosphatases (PPs) or by Tyr-phosphatases (PTPs). The mechanism of dephosphorylation of the MAPKs ERK2 and p38α by PTPs is favoured by docking interaction between the PTP and the MAPK. However, how dephosphorylation of MAPKs by PPs is regulated, and how PTPs and PPs cooperate in MAPK inactivation, is not well understood. In this work, we show a differential role of the Tyr at the MAPK Thr-X-Tyr motif in the inactivation of ERK2 and p38α by phosphatases. Inhibition of the dephosphorylation of the activation loop-Tyr residue, either by disrupting the complex MAPK-phosphatase with a MAPK docking site mutation or by using the PTP inhibitor sodium orthovanadate, impaired the dephosphorylation by PPs of the ERK2-Thr residue, but not of the p38α-Thr residue. Conversely, the dephosphorylation by the PTPs PTP-SL and HePTP of the ERK2 activation loop-Tyr residue promoted the dephosphorylation of the Thr residue by Ser/Thr-phosphatases sensitive to okadaic acid. Our results indicate that the dephosphorylation by PPs of the activation loop-Thr residue from ERK2 and p38α, differentially depends on the previous Tyr-dephosphorylation by PTPs, and suggest ERK2- and p38α-specific models of MAPK inactivation by sequential or cooperative action of PTPs and PPs.