The sequence of the vaccinia virus open reading frame F2L predicts a polypeptide with significant similarity to cellular dUTPases. To determine whether the F2L gene product has this activity, it was expressed in bacteria as a fusion with glutathione S-transferase. Affinity purified fusion protein was shown to hydrolyze dUTP yielding dUMP as the product. While the dUTPase was not completely dependent on the addition of divalent cations, its activity was stimulated markedly by Zn2+, Mg2+, and Mn2+. The nucleotide substrate specificity of the enzyme was limited to dUTP. These results demonstrate that vaccinia virus encodes a functional dUTPase whose role in viral infection is suggested to be the augmentation of DNA nucleotide precursors and the minimization of cytoplasmic dUTP concentrations.