MAP kinases are involved in numerous signaling processes and are crucial for normal function of cells and organisms. MAP kinases are mainly activated via the canonic three-component cascade leading to dual phosphorylation on the adjacent Thr-180 and Tyr-182 (p38 numbering) located in the phosphorylation lip. Several alternative activation pathways and modes have been shown to occur in p38a MAP kinase. One of these is induced by T-cell receptor activation and subsequent phosphorylation of p38a on the distinctive Tyr-323 distal from the phosphorylation lip, by ZAP-70 tyrosine kinase. Consequent to Tyr-323 phosphorylation, autoactivation in trans occurs, resulting in mono-phosphorylation of Thr-180. This alternative pathway differs in its substrate selectivity profile from the canonic one, and elucidating its structure would allow insight into this enigmatic active form and its mode of function. Due to the lack of phosphorylated Tyr-323 (pTyr-323) for structural studies, we elected to find active mutants that emulate the functional role of pTyr-323. Several Tyr-323 point mutations exhibiting intrinsic activity and autophosphorylation capabilities were identified. We assumed that these active mutants could mimic the conformational changes induced by pTyr-323 and thus reveal its unique activation mechanism. These changes were evaluated by determining the X-ray structures of selected active (Y3232Q/T/R) and inactive (Y323F) mutants. Structural analysis revealed, for the first time, dramatic conformational changes in p38a, capturing the distinctive factors promoting autoactivation. In this regard, the active mutants induce dramatic changes in the kinase interlobe orientation, which with the unique conformation of the activation loop contribute to the formation of the additional substrate docking DEF site interaction pocket. These combined structural features make p38 receptive for trans autophosphorylation on Thr-180. These mutants could become a powerful tool for understanding the alternative activation pathway exclusive to T cells and the differences in the resulting phenotypes.