The pyrimidine (uracil) analogue 3-oxauracil (OU) previously had been shown to completely inhibit the growth of E. coli B and decrease by 96% the replication of herpes simplex virus type 2 when present in the culture fluid at a concentration of 10(2) microM. Limited in vivo studies in mice demonstrated antiviral effects without significant toxicity when given i.p. daily for two weeks at a concentration of 3.23 mg/kg. However, the antineoplastic properties of OU were unknown. We assessed the ability of OU to inhibit the proliferation of various human tumor cell lines (3 pancreatic, 1 colon, 1 neuroendocrine, and 1 lung) in an in vitro radiometric (Bactec) system. In the pancreatic lines (RWP-2, MiaPaCa-2, and PANC-1), the colon line (HT-29), the neuroendocrine line (COLO 320DM), and the lung cancer cell line (SK-MES-1), OU at a concentration of 10(3) microM, produced a dramatic decrease in percent cell survival. When compared with cytotoxic drugs of choice for these tumor cells (gemcitabine, 5-fluorouracil, and adriamycin, respectively) a significantly higher concentration of OU was required usually to achieve comparable results with two exceptions. These were the HT-29 and the COLO 320DM cell lines. These results indicate OU has significant (p < 0.05) cytotoxic activity against pancreatic, colon, neuroendocrine, and nonsmall cell lung cancer lines, when compared to untreated control cultures. Additional in vivo testing of this potential antineoplastic agent is warranted.