Eukaryotic initiation factor 2B, or guanine nucleotide-exchange factor, has been purified for the first time from the brain by a novel procedure that allows the purification of initiation factor 2 as well and uses a salt wash postmicrosomal supernatant as starting material. The procedure includes a three-part chromatographic step in heparin-Sepharose and in SP-5PW and diethylaminoethyl-5PW ion-exchange high-performance chromatographies. The purification of the factors was followed by measuring activity in the guanine nucleotide-exchange assay and the capacity of initiation factor 2 to form a ternary complex with the initiation form of methionyl-tRNA and GTP. The method yields guanine nucleotide-exchange factor (75%) and highly purified initiation factor 2 (> 95%), which are separated in the last step. The exchange factor from the brain is a multimeric protein with five subunits of molecular masses of 82, 65, 52, 42, and 30 kDa; it stimulates ternary complex formation in the presence of GDP, and this activity is inhibited by N-ethylmaleimide. A 37-kDa protein that copurifies with initiation factors is characterized in this study as a new calmodulin-binding protein (p37); it is highly phosphorylated by casein kinase activities and can comigrate with the alpha subunit of initiation factor 2 under standard sodium dodecyl sulfate electrophoresis conditions.