A recombinant plasmid, pHW1, directing the overproduction of the enzyme deoxyuridine 5'-triphosphate nucleotidohydrolase (dUTPase, EC 3.6.1.23) from Escherichia coli has been constructed. A 1900-base DNA fragment carrying the structural gene for the enzyme (dut) has been recloned into a runaway replication vector that also carries the strong leftward promoter (pL) of bacteriophage lambda. Upon temperature shift, an E. coli strain carrying the new plasmid gives an increase in dUTPase activity of about 600-fold in rich medium compared to wild-type bacteria. The 64-kDa protein corresponding to the mature form of the enzyme reaches 20% of the total protein content of the bacterial cell. Using this strain, a simplified procedure has been developed for the purification of dUTPase. The purification steps consist of extraction of the cytoplasmic proteins, ammonium sulfate precipitation, anion-exchange chromatography and gel filtration on FPLC. The new overproducing plasmid and the simplified purification procedure developed will make it possible to purify dUTPase in sufficient amounts for detailed characterization studies.