The photodegradation of the chemotherapeutic agent 5-fluorouracil (5-FU) under UVB light was studied both in aqueous and methanol solutions and in systemic and topical formulations. As monitored by HPLC, photodegradation in solution takes place in a concentration dependent manner; thus, the solution for parenteral administration (10(-1) M) showed negligible loss of the active principle. On the contrary, the commercial cream containing 5% of 5-FU showed low stability under UVB exposure. When dissolved either in water or methanol, 5-FU yields two photoproducts which have been characterized as two isomers coming from the addition of the solvent to the 5,6 double bond of the drug. As a consequence, photomodified 5-FU loses its antiproliferative activity on HCT-15 and HeLa cells. MS analysis showed that photoaddition occurred with nucleophilic amino acids, such as cysteine and serine, while susceptible amino acids (cysteine and methionine) were oxidized. In fact, high production of the superoxide anion under UVB light as well as photooxidation of BSA suggests protein photodamage as a mechanism of photosensitization. Indeed, some phototoxicity was shown in experiments on NCTC keratinocytes and MCF-7 resistant cells irradiated with UVB light. The interactions with these biological targets may contribute to skin phototoxicity and photoallergy induced by 5-FU in vivo.