Addition of selenite to rabbit reticulocyte lysate produces a biphasic pattern of translational inhibition. Sucrose density gradient shows that the onset of translational inhibition is accompanied by decreased Met-tRNAf binding to 43 SN ribosomal subunits and loss of polysomes. Control rates of translation are restored by the addition of exogenous eukaryotic initiation factor 2 (eIF-2). Selenite also directly inhibits Met-tRNAf binding activity of eIF-2. While selenite could react directly with unpaired cysteine residues of eIF-2 to inhibit protein synthesis initiation, a more complex mechanism than a direct inactivation of eIF-2 is suggested by the following observations: 1) translational inhibition produced by selenite is accompanied by an apparent increase in the phosphorylation state of eIF-2alpha; and 2) the extent of translational inhibiton is not proportional to steady-state level of phosphorylation. Rather, the time required for the onset of translational inhibition decreases as the level of eIF-2alpha phosphorylation is increased. This suggests a multistep sequence for eIF-2 inactivation, dependent upon an initial activation of eIF-2alpha kinase and followed by additional eIF-2 modification(s).