Because the phosphorylation of non-histone proteins has been suggested to play a role in the regulation of eukaryotic gene transcription, we have compared the phosphorylation of these proteins in normal and SV-40 transformed WI-38 human diploid fibroblasts. The rate of phosphorylation was found to be roughly ten-fold higher in the transformed cells, and this striking difference could not be accounted for by either an increased rate of phosphate transport or by the synthesis of new species of non-histone proteins which subsequently become phosphorylated. To our knowledge this is the most dramatic alteration in non-histone protein phosphorylation ever described, and therefore may have important implications for our understanding of malignant transformation.