Treatment of eIF-2B and eIF-2 with NEM abolishes nucleotide exchange and GTP-binding activities of the proteins. Incubation of eIF-2B with [14C]NEM results in strong labeling of the 82- and 55-kDa subunits and with less labeling of the other subunits. Preincubation of eIF-2B with eIF-2 interferes with [14C]NEM labeling of the 82- and 55-kDa subunits. All three (α,β,and γ) subunits of eIF-2 are labeled strongly by [14C]NEM. Limited digestion of eIF-2B with trypsin inhibits nucleotide exchange activity but does not interfere with GTP binding. Under these conditions, the 65-kDa subunit is degraded completely while the other subunits remain intact. Treatment of eIF-2 with trypsin results in the generation of eIF-2 lacking the β-subunit (eIF-2αγ). eIF-2(αγ) binds [3H]GDP equally well as intact eIF-2. In the presence of eIF-2B, the exchange of [3H]GDP for GTP from eIF-2.[3H]GDP prepared with eIF-2(αγ) is diminished considerably. [3H]GTP binding to eIF-2 (αγ) is also four- to five-fold less than to intact eIF-2. In addition, the association of eIF-2B with intact eIF-2, but not with eIF-2(αγ), reduces by two-fold the rate and extent of removal of32P by alkaline phosphatase from CK-2-phosphorylated 82-kDa subunit.