The eukaryotic protein synthesis initiation factor, eIF‐2B, is a multimeric protein of five different subunits termed α, β, γ, δ and ɛ, which facilitates recycling of a further factor, eIF‐2, and is an important control point in the initiation process. In order to investigate the structure and function of eIF‐2B, monoclonal antibodies have been prepared to the β, δ and ɛ subunits of the factor from rabbit reticulocytes. All three antibodies are active in Western blotting, ELISA and immunoprecipitation. The anti‐ɛ antibody inhibits both the guanine nucleotide exchange activity of eIF‐2B and protein synthesis in the rabbit reticulocyte lysate at the level of initiation. The other two antibodies do not inhibit either guanine nucleotide exchange or protein synthesis. The monoclonal antibodies and a polyclonal anti‐(rabbit reticulocyte eIF‐2B) serum were used to investigate the subunit size and the antigenic structure of eIF‐2B from a variety of rabbit tissues and from a variety of mammalian species. eIF‐2B from all rabbit tissues tested was indistinguishable from that prepared from rabbit reticulocytes. Quantitative studies showed substantial variation in the relative concentrations of eIF‐2 and eIF‐2B between different rabbit tissues. Marked variation in both the sizes of the subunits and their reaction with the antibodies was observed between eIF‐2B from rabbit, rat, guinea pig and man.