Translational rates, and activities and levels of initiation factors 2 and 2B were assessed in rat pheochromocytoma cells upon nerve growth factor (NGF) treatment. Two or 5 days of exposure to NGF caused significant quantitative increases in protein synthesis rate that are deemed necessary for neuronal differentiation. Changes in initiation factor 2 activity, as measured by its capacity to form a ternary complex, occur parallel to the observed changes in protein synthesis. Nevertheless, neither the intracellular levels of the initiation factor 2 nor the degree of phosphorylation of its alpha subunit can justify this increased activity. Interestingly, initiation factor 2B activity increases parallel to the neurite outgrowth, being significantly higher after 5 days of exposure to NGF, and could be responsible for the elevated rate of protein synthesis. No significant changes in the levels of eukaryotic initiation factor 2B, as determined with two different antibodies against the gamma and epsilon subunits of the factor, were observed, implying that the increased activity should be regulated by factors other than its cellular concentration. Our results support the hypothesis that initiation factor 2B may play a role in the biochemical events controlling the differentiative growth factor-induced signaling pathway in these cells.