To determine the relationship between 5-fluorouracil (FUra) toxicity and its RNA- and DNA-directed actions we examined the ability of continuous SC infusions with uridine (Urd), thymidine (dThd), or deoxyuridine (dUrd) to rescue mice from the lethal toxicity of FUra. Male B6D2F1 mice were treated with a single IP injection of FUra (800 mg/kg) followed in 24 h by a 5-day infusion with either 0.9% NaCl or Urd (0.1, 1, 5, or 10 g/kg/day). Survivors were then followed up for 30 days after FUra treatment. Urd (1, 5, or 10 g/kg/day) rescued mice from the lethal toxicity of FUra, whereas Urd (0.1 g/kg/day) was as ineffective as 0.9% NaCl as a rescue agent. With variable doses of FUra followed in 24 h by a Urd infusion (5 g/kg/day) for 5 days. Urd rescued mice treated with FUra (400, 600, or 800 mg/kg) but was ineffective against higher doses of FUra (1,000 or 1,200 mg/kg). Mice treated with FUra (800 mg/kg) followed in 24 h by a 5-day infusion with either dThd (1, 5, or 10 g/kg/day) or a dUrd (1 or 5 g/kg/day) could not be rescued from the lethal toxicity of FUra. In all experiments deaths occurred between 6 and 12 days after FUra. These results, which demonstrate a specificity for Urd, but not for either dThd or dUrd, for rescuing mice from the lethal toxicity of FUra, suggest the importance of the RNA- rather than the DNA-directed actions of FUra as a determinant of its toxicity in mice.