Recent studies from several laboratories have suggested that the anticancer drug-5-fluorouracil (FUra) promotes abnormal splicing of precursor RNA molecules. In order to determine the effects of FUra on the chemistry of RNA splicing, we studied the splicing reaction of FUra-containing Tetrahymena rRNA [(FUra) RNA], a Group I self-splicing system having one intron [intervening sequence (IVS)] and two exons. When subjected to splicing conditions, the (FUra) precursor RNA gave all of the normal splicing products, ligated exons, IVS, circulation IVS (C-IVS), and the hydrolyzed circle (L-19 IVS) as well as other hydrolysis side products. No abnormal products indicative of missplicing were observed at pH 7.5. However, the presence of FUra in the RNA decreased the rates and extents of formation of all of the product species. At pH 7.5, the rate of ligated exon formation of (FUra) RNA was inhibited 3-fold and the maximum yield of ligated exons was 50% of normal. Substitution with FUra inhibited the rate of formation of C-IVS about 2-fold, while the extent of formation of this product was decreased by more than 3-fold compared to uracil-containing RNA [(Ura) RNA]. The circularization of (Ura) IVS remained constant to pH 9 and then increased, while that of (FUra) IVS declined abruptly after pH 7.3, indicating that ionization of the FUra residues of (FUra) RNA abolishes its catalytic activity. A temperature dependence experiment showed that the circularization activity of (FUra) IVS was lost at a temperature 15 degrees C lower than that of the (Ura) IVS. The labile phosphodiester bond of the (FUra) C-IVS was more stable to hydrolysis than was that of the (Ura) C-IVS at all pH values. The data suggest that a major effect of FUra substitution is to destabilize the active conformation of RNA because of weaker base pairing between FUra and adenine owing to partial ionization of the FUra residues.