The catalytic activity of many kinases is regulated by the phosphorylation/dephosphorylation of specific Ser, Thr or Tyr residues located at their activation loop. In the case of the classical MAP kinase (MAPK) family (ERK1/2, ERK5, p38s and JNKs), the catalytic activity depends on the phosphorylation status of a Thr and a Tyr that are separated by an unique and variable residue (Glu for ERK1/2 and ERK5, Gly for p38s, and Pro for JNKs). Specific MAPK phosphatases, including Tyr-phosphatases (PTPs) and dual specific-phosphatases (DSPs), dephosphorylate the Thr-X-Tyr motif and inactivate MAPKs. We have observed that JNK1 is more resistant to inactivation by phosphatases in cell extracts than ERK2 and p38α. In accordance, inactivation of JNK1 in intact cells was delayed when compared to p38α. To investigate the influence of the variable residue at the MAPK Thr-X-Tyr motif in the inactivation of these enzymes, we have interchanged this residue between ERK2, p38α, and JNK1, and analyzed the dephosphorylation of the wild type and mutated activation loops in cell extracts and in intact cells. Our results indicate that: 1/ the Glu residue from ERK2 activation motif is important for the efficient dephosphorylation of the MAPK; 2/ the Gly residue from p38α activation motif is dispensable for the dephosphorylation and inactivation of p38α; and 3/ the Pro residue from JNK1 activation motif hampers JNK1 dephosphorylation and inactivation. We conclude that the variable residue at the MAPK Thr-X-Tyr motif confers specificity to each MAPK in their regulation by phosphatases.