In heme deficiency, protein synthesis is inhibited by the activation of the heme-regulated eIF2alpha kinase (HRI) through its multiple autophosphorylation. Autophosphorylation sites in HRI were identified in order to investigate their functions. We found that there were eight major tryptic phosphopeptides of HRI activated in heme deficiency. In this report we focused on the role of autophosphorylation at Thr483 and Thr485 in the activation loop of HRI. Disruption of the autophosphorylation of Thr485, but not Thr483, resulted in a lower autokinase activity and locked Thr485Ala HRI in a hypophosphorylated state. Most importantly, autophosphorylation of Thr485, but not Thr483, was essential for attaining eIF2alpha kinase activity of HRI. In addition, autophosphorylation of Thr485 was necessary for arsenite-induced activation of the eIF2alpha kinase activity of HRI, while autophosphorylation at Thr483 was not required for activation by arsenite. The function of Thr490, another conserved Thr residue in the activation loop of HRI, was also investigated. Mutations of Thr490 to either Ala or Asp resulted in reduced autokinase activity and loss of eIF2alpha kinase activity in heme deficiency or upon arsenite treatment. Since Thr490 was not identified as an autophosphorylated site, it is likely that Thr490 itself might be critical for the catalytic activity of HRI. Importantly, Thr485 was very poorly phosphorylated in Thr490 mutant HRI. Collectively, our results demonstrate that autophosphorylation of Thr485 is essential for the hyperphosphorylation and activation of HRI and is required for the acquisition of the eIF2alpha kinase activity.