Hyperoxia is cytotoxic and depresses many cellular metabolic functions including protein synthesis. Translational control is exerted primarily during initiation by two mechanisms: 1) through inhibition of translation initiation complex formation via sequestration of the cap-binding protein, eukaryotic initiation factor (eIF) 4E, with inhibitory 4E-binding proteins (4E-BP); and 2) by prevention of eIF2^.GTP^. tRNA_i ^Met formation and eIF2B activity by phosphorylated eIF2. In this report, exposure of human lung fibroblasts to 95%O₂ decreased the incorporation of thymidine into DNA at 6 hours and the incorporation of leucine into protein beginning at 12 hours. The reductions in DNA and protein synthesis were accompanied by increased phosphorylation of eIF4E protein and reduced phosphorylation of 4E-BP1. At 24 hours, hyperoxia shifted 4E-BP1 phosphorylation to lesser-phosphorylated isoforms, increased eIF4E expression, and increased the association of eIF4E with 4E-BP1. Although hyperoxia did not change eIF2 expression, it increased its phosphorylation at Ser-51, but not until 48 hours. In addition, the activation of eIF2 was not accompanied by the formation of stress granules. These findings suggest that hyperoxia diminishes protein synthesis by increasing eIF4E phosphorylation and enhancing the affinity of 4E-BP1 for eIF4E.