Eukaryotic initiation factor eIF2B plays a key role in the regulation of protein synthesis through its ability to catalyze the exchange of GDP bound to a second initiation factor, eIF2, for free GTP. In contrast to other GDP-GTP exchange factors (GEFs), which are often single subunit proteins, eIF2B consists of five dissimilar subunits. In the studies reported here the baculovirus expression vector system (BEVS) was used to express FLAG epitope tagged alleles for the alpha, beta, gamma, delta, and epsilon subunits of rat eIF2B in Sf21 cells. The eIF2B holoprotein was reconstituted in vivo by coexpression of all five subunits in Sf21 cells and was subsequently purified to greater than 98% homogeneity using a two-step procedure involving an anti-FLAG immunoaffinity column followed by gel filtration chromatography. The purified five-subunit eIF2B complex had high GEF activity as assayed by using [3H]GDP-bound to eIF2 as a substrate. Alternatively, eIF2B with high GEF activity was reconstituted in vitro by mixing crude cell lysates containing different eIF2B subunits. The latter results suggest that eIF2B activity in vivo could involve alterations in the concentration and/or the availability of individual subunits for holoprotein assembly. Overall, the results show the utility of the baculovirus-insect cell system for the expression, assembly, and purification of active recombinant multisubunit factors.