Four 5-alkynyl-2′-deoxyuridines containing different bulky substituents and flexible linkers between the triple bond and the aromatic residue have been prepared and tested against HSV-1 in Vero cells. Two nucleosides containing carbonyl groups, 5-(4-benzoylphenoxypropyn-1-yl)- 2′-deoxyuridine (19a) and 5-(estron-3-yloxypropyn-1-yl)-2′- deoxyuridine (19c), showed low cytotoxicity and moderate antiviral activity. The flexible linker appears not to be favorable for antiviral properties of 5-alkynyl-2′deoxyuridines: 5-[(perylen-3-yl)methoxypropyn-1-yl]-2′- deoxyuridine (19d) showed considerable cytotoxicity and no antiviral activity in contrast to the active and nontoxic 5-(perylen-3-ylethynyl)-2′- deoxyuridine (9), a nucleoside with a rigid triple-bond-connection of the aromatic system to the nucleobase. © 2005 Elsevier Ltd. All rights reserved.