Jak2 is a member of the Janus family of tyrosine kinases and is known to be activated by a wide variety of ligands. Here, we sought to identify amino acid residues within Jak2 that are essential for its activation. We provide evidence that glutamic acid 1046 (E1046) is one such residue. Using molecular modeling algorithms of the Jak2 kinase domain, we identified a putative molecular interaction between E1046 and tryptophan 1020 (W1020). Conversion of E1046 to either arginine (E1046R), alanine (E1046A), aspartic acid (E1046D) or glutamine (E1046Q) abolished Jak2 kinase activity as measured by autophosphorylation assays. Conversion of W1020 to glycine (W1020G) similarly abolished Jak2 kinase activity. Finally, we tested the ability of the E1046R mutant to activate the Jak/STAT signaling pathway in a ligand-dependent signaling system. The ability of angiotensin II to activate the Jak/STAT signaling pathway in cells expressing the E1046R mutant was severely compromised as measured by reduced (1) Jak2 autophosphorylation (2) Jak2 kinase activity (3) AT₁/Jak2 co-association (4) Stat1 tyrosine phosphorylation and (5) angiotensin II-mediated gene transcription. Thus, these studies demonstrate for the first time, the critical role of E1046 in mediating Jak2 activation and its subsequent downstream signaling events.