The antiproliferative activity screening on human tumor cell lines of a series of modified uracil and cytosine bases as well as some corresponding acyclonucleosides, and comparison of structure-activity relationship revealed the importance of chemical reactivity of the substituent attached to the C5-position of uracil for the activity of studied compounds. Namely, the results obtained for the most active compounds, 5-(chloroacetylamino)uracil (2) and its acyclic sugar analogue 18, suggest that formation of a covalent bond between reactive substituent and several possible targets within the thymidylate synthase mechanism (sulphur of the cysteine residue, basic part of the enzyme, N,N-methylene tetrahydrofolate or its reactive iminium forms) is the most probable mode of action. In addition, novel C5-substituted uracil derivative 6 (5-[bis-(2-p-methoxybenzylthioethyl)amine]acetylaminouracil) exhibited high antiproliferative activity against HeLa and MiaPaCa-2 cell lines, by an as yet unknown mechanism.