Nascent DNA (nDNA) replication intermediates can be isolated and quantified by pH-step alkaline elution (L.C. Erikson et al,, Chromosoma, 74, 125-139, 1979). The effects of 5-fluorouracil (FU) and 5-fluoro-2'-deoxyuridine (FdU) on the formation and persistence of nascent DNA structures were studied in Chinese hamster ovary K1 cells. Exogenous thymidine (dT) rescued FdU-induced, but had little effect on FU-induced, cytotoxicity, indicating DNA- and RNA-directed cytotoxic mechanisms respectively. Drug-treated cells were pulse labelled with [H-3]dT and the percentage of total counts eluting from and retained by filters estimated at successive pH increments (11.0, 11.3, 11.5 and 12.1). A 1 min pulse of control cells resulted in >75% of the DNA eluting at pH 12.1 or being retained by the filter. By 1 h, >90% of the DNA was retained by the filter. FdU treatment resulted in the persistence of DNA in all four pH bands. More than 95% of total DNA synthesized during a 5 min pulse eluted as discrete peaks at each pH, This DNA was processed to higher M(r) species during a 1 h chase, but following a 24 h chase, 68% of the DNA still eluted at pH 12.1. In contrast, FU treatment caused only a transient accumulation (less than or equal to 10 min) of the DNA (e.g. similar to 26% at 5 min) in the pH 11.0 and 11.3 bands only, indicating a selective effect of FU on the maturation of very short size classes of DNA. Neutral elution was used to assess the effect of FdU on double-strand break (dsb) formation in nDNA. Whereas no dsbs were evident in untreated cells, dsbs appeared in FdU-treated cells even following the shortest [H-3]dT pulse times (1-10 min). Although these were processed into higher M(r) species with longer pulse times, dsbs were still evident at 2 h.