Insulin increases the rate of overall protein synthesis in many cells and tissues, while inducing the preferential expression of individual proteins. To identify and characterize such proteins, NIH 3T3 cells stably expressing more than 10(6) human insulin receptors per cell (HIR 3.5; Whittaker, J., Okamoto, A. K., Thys, R., Bell, G. I., Steiner, D. F., and Hofmann, C. A. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 5237-5241) were treated with insulin in the presence of [35S]methionine, and labeled proteins were separated using ultra-high resolution ("giant") two-dimensional gel electrophoresis. Overall protein synthesis was enhanced as much as 3-fold by insulin treatment; the synthesis of approximately 1% of the 2,500 proteins visible on the gel autoradiographs was further selectively increased. By using immunoblotting, immunoprecipitation and comigration assays, we identified one of the rapidly induced proteins (Mr 96,000; pI 6.8) as eukaryotic elongation factor 2 (EF-2), a major component of the protein translation apparatus. Insulin induced the synthesis of EF-2 within 20 min of treatment, with a half-maximal dose of 10(-11) M. It was synthesized as a precursor form that was processed to a more basic mature species within 30 min. Long term treatment with insulin led to accumulation of EF-2 within the cell and prevented the substantial decrease in EF-2 concentration that occurred during serum deprivation. Finally, we found that insulin induction of EF-2 occurred normally in the presence of the RNA-transcription inhibitor, actinomycin D. Thus, insulin rapidly induced the synthesis of EF-2 predominantly or exclusively at the level of mRNA translation.