Current evidence suggests that DNA fragmentation plays an integral role in mediating cytotoxicity that results from thymidine nucleotide depletion ("thymineless death"). Recently, Ayusawa et al. [Mutat. Res. 200:221-230 (1988)] reported that dTMP starvation induces cellular processes that result in the release of 50-200-kilobase (kb) DNA fragments in FM3A cells, as detected by pulsed field gel electrophoresis (PFGE). The present study was undertaken to determine whether a similar DNA fragmentation process occurs in a human cell line in response to fluoropyrimidine treatment and, if so, to quantitate this process. When human colorectal tumor (HT29) cells were treated with 100 nm 5-fluorodeoxyuridine (FdUrd), this regimen induced the formation of high molecular weight DNA fragments, which were analyzed using three different PFGE protocols. Field inversion PFGE revealed that, in contrast to the discrete size range reported for FM3A cells, FdUrd-induced fragments in HT29 cells were broadly distributed from about 50 kb to sizes beyond the resolution of the field inversion mode (i.e., greater than 600 kb). Analysis of these same samples by contour-clamped homogeneous electric field PFGE showed that the bulk of these fragments migrated in the 1-5-megabase region. In contrast, fragments from DNA that was broken randomly by gamma-radiation appeared to be primarily in a zone corresponding to approximately 5-10 megabases. Equitoxic FdUrd and radiation treatments (100 nM FdUrd for 48 hr versus 10-Gy gamma-radiation) each increased the fraction of DNA entering the gel from about 0.07-0.09 (untreated cells) to about 0.22-0.25. To a first approximation, the time course and quantity of DNA fragmentation induced by 100 nM FdUrd appeared to correlate with the loss of clonogenicity within the 48-hr period analyzed. These findings suggest that the processes responsible for DNA fragmentation in response to a thymineless state may be different in FM3A and HT29 cells, that in both cases the breaks caused do not appear to be located randomly with respect to the entire genome, and that these processes may be related to the chain of events by which temporary dTMP starvation is made into a lethal insult.