Quantitative S1 nuclease mapping studies were performed with uniformly labeled RNA probes, containing contiguous dihydrofolate reductase exon and intron sequences, and total RNA isolated from KB7B cells exposed to 5-fluorouracil for 5 days. Dihydrofolate reductase RNA containing both exon 1 and intron I, or exon 5 and a portion of intron V, increased up to 5-fold in cells grown in the presence of 2.0 to 3.0 microM 5-fluorouracil. Dihydrofolate reductase RNA containing exon 1 or exon 5, but lacking intron I or intron V, respectively, increased 2-fold in cells grown in the presence of 0.65 to 3.0 microM 5-fluorouracil. Primer extension analysis and S1 mapping studies revealed two major transcriptional start sites at positions -72 and -69 and minor start sites upstream from position -183, for dihydrofolate reductase RNA isolated from methotrexate-resistant KB7B cells. The results of these studies demonstrate that 5-fluorouracil alters the metabolism of dihydrofolate reductase precursor mRNA and/or processing intermediates.