The biosynthesis and phosphorylation of nuclear proteins of thymocytes were investigated in rats after 4.0 Gy whole-body X-irradiation during the period which precedes DNA degradation in lymphoid cells. Proteins were separated by two-dimensional gel-electrophoresis, detected by Coomassie blue staining. Incorporation of [35S]methionine into proteins and phosphorylation of proteins with [32P]inorganic phosphate were determined by scanning densitometry of autofluorograms and autoradiograms, respectively. No change in the quantity of proteins was observed 1 h after irradiation. Decrease in specific activity of [35S]methionine-labelled proteins was seen in most protein fractions. Significant enhancement of phosphorylation of three proteins was established, characterized by molecular weight and pH: MW 20 kD, pH 6.8; MW 35 kD, pH 5.8 and MW 48 kD, pH 5.8. These results suggest that immediately after X-irradiation a short-term increase of chromatin-bound non-histone protein phosphorylation occurs. This finding, along with the previously described enhancement of RNA polymerase II in thymocytes (Zhivotovsky et al. 1982) suggests a temporary gene activation shortly after X-irradiation of the rat.