We have developed a database of lymphoid polypeptides detected by two-dimensional polyacrylamide gel electrophoresis to aid in studies of leukemogenesis and of mutation affecting protein structure. In prior studies, we observed a 19-kDa phosphopolypeptide which was induced with proliferation in mature T cells and constitutively expressed in immature thymocytes. In this report we describe the identification of this polypeptide as the phosphorylated form of dUTPase (EC 3.6.1.23), following cDNA cloning of the gene, based on a partial amino acid sequence of the phosphopolypeptide. Studies of the expression and phosphorylation of dUTPase in human T cells indicate that accumulation and phosphorylation of dUTPase in mature T cells occur in a cell cycle-dependent manner. Interestingly, noncycling immature thymocytes express constitutively high levels of phosphorylated and unphosphorylated dUTPase. These results suggest an important role for dUTPase in immature thymocytes that is independent of proliferation.