In vitro exposure of a human lymphoblastoid cell line (WIL-2) to the antifolate metoprine (DDMP), when followed by the addition of exogenous deoxyuridine, led to intracellular accumulation of deoxyuridine triphosphate (dUTP) and incorporation of deoxyuridine monophosphate (dUMP) into DNA.When newly synthesized DNA was extracted from DDMP-treated cells that had been labeled with deoxyuridine for up to 3 min, most of the DNA synthesized was no larger than 4 Prokaryotic and eukaryotic cells contain a potent deoxyuridine triphosphatase (1-4) and a uracil N-glycosylase-dependent repair system (5-9) that normally prevent accumulation of dUMPcontaining DNA. We have found that, when intact WIL-2 human lymphoblastoid cells are inhibited by antifolates methotrexate (MTX) or metoprine [2,4-diamino-5-(3',4'-dichlorophenyl)-6-methylpyrimidine, DDMP], the apparent levels of intracellular dUMP can be proportionally increased by exposure to increasing exogenous concentrations of deoxyuridine (10). In this study we examined the fate of exogenously added deoxyuridine in DDMP-treated cells, in order to determine whether dUTP becomes detectable and leads to dUMP misincorporation. We also looked for perturbations of DNA synthesis that might occur as a result of this lesion. Specifically, it was important to determine whether antifolates and deoxyuridine might affect the normal progression of DNA synthesis and not simply decrease overall incorporation into DNA products of all sizes. For rapid pulse experiments, cells were injected directly into 60% methanol at -20°C. Extraction of nucleosides and nucleotides was carried out for at Itast 24 hr before further processing of the samples. MATERIALS AND METHODS Chemicals andNucleotide Pool Analysis by Cellulose Thin-Layer Chromatography. The methanol nucleotide pool extracts described above were lyophilized to dryness and resuspended in 30 ,ul of 10% isopropyl alcohol/10% acetic acid/80% water (vol/vol).DNA nucleotides (0.01 M) were used as standards for chromatography. Cellulose plastic plates (Kodak), impregnated with fluorescent dye indicator, were divided into 10 numbered channels. Portions of each sample were spotted at the origin, positioned at 2 cm from the bottom of the plate for each channel. Portions of standard solutions were spotted together with or beside each sample, and the position of migration was visualized by irradiating the plates with shortwave ultraviolet light. The solvent system utilized for the nucleotide separations was isobutyric acid/NH40H/H20(63:1:33 vol/vol). Thin-layer chromatography plates were developed for approximately 4 hr (or until the solvent had moved 17 cm from the bottom of the plate) Abbreviations: MTX, methotrexate; DDMP, 2,4-diamino-5-(3',4'-dichlorophenyl)-&methylpyrimidine (metoprine); PEI, polyethyleneimine. 917The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.