Several N-substituted derivatives of 5-fluorouracil have been synthesized during the development of new antitumor agents. For determination of the position of the substituent on these compounds, electron impact mass spectrometry was investigated. In their electron impact mass spectra, characteristic fragment ions were produced by the retro Diels--Alder decomposition of the 5-fluorouracil skeleton: [R(or R')N=CHC(F)=C=O]+. was produced from N-1 substituted derivatives, while an isocyanate ion, [RN=C=O]+., or an acylurea ion, [RCONHCONH2]+. was formed from N-3 substituted derivatives. The ions observed were useful for structural determination.