In Ehrlich ascites tumor cells maintained in serum-free medium for 16 h the rate of protein synthesis was about 50% of the rate in control (well-fed) cells. The addition of 10% calf serum led to a 1.5- to 2-fold stimulation of protein synthesis within 10 min. Stimulation was effected through a non-transcriptional mechanism which operated at the level of polypeptide chain initiation. The effect was due to non-dialyzable serum growth factors which were sensitive to treatment with dithiothreitol and iodoacetamide. Replacing the 16-h-conditioned serum-free medium with fresh serum-free medium stimulated protein synthesis about 30% in serum-deprived cells, and the effect of these low molecular weight nutrients was additive with the effect of serum factors. Phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF-2 alpha) inhibits protein synthesis by competitively inhibiting the guanine nucleotide exchange factor (GEF), and modulation of the extent of phosphorylation of eIF-2 alpha has been suggested as a probable regulatory mechanism in serum-deprived mammalian cells. We measured the ratio of phosphorylated to total eIF-2 alpha in serum-deprived cells. The ratio was elevated in serum-deprived cells compared to control (serum-fed) cells. eIF-2 was rapidly dephosphorylated in response to serum refeeding and returned to near control levels after 10 min. The rapidity of this response and the close temporal correlation between eIF-2 dephosphorylation and increased rate of protein synthesis provide evidence that eIF-2 plays an important role in the regulation of protein synthesis by serum growth factors.