5-Fluorouracil (5-FU) is one of the most widely used antineoplastic drugs. It can be therefore considered to be a model compound for the identification of exposure routes during preparation and administration of cytostatic agents, especially for nucleoside analogue drugs. In this study, an HPLC-UV method was validated for determination of 5-FU in wipe samples by direct analysis of the aqueous solutions and in air samples by using solid-phase extraction (SPE). When samples were pre-treated on styrene-divinylbenzene resin SPE columns, a 20-fold preconcentration of the analyte was achieved. As regards air samples, correlation coefficients were always higher than 0.998 and the limit of detection was assessed at 15 ng on filter. In order to verify the reliability of these procedures, 5-chlorouracil was used as internal standard. The procedure presented here has been applied to the environmental monitoring of occupational exposed subjects. The amount of 5-FU ranged from 0.043 to 0.23 microg/m3 in air samples and from 0.2 to 470.1 microg/dm2 in wipe samples. 5-FU was also detected on the internal side of the gloves (0.07 to 3.77 microg/pair of gloves).