A series of 1,3-dioxolanyluracil analogues was prepared from the dioxolane intermediates 2, and their anti-Epstein Barr virus (anti-EBV) activities were determined. The potency of L-dioxolane uracil nucleosides against EBV replication is dependent on the substituents at the 5-position in the following decreasing order: I > Br > Cl > CH3 > CF3 > F. The most active and selective analogue was the iodo derivative (L-I-OddU) with an EC50 value of 0.03 microM and an EC90 value of 0.16 microM. There was no cytotoxicity or depletion of mitochondrial DNA in cells after exposure to L-I-OddU at 50 microM. The action against EBV replication in H1 cells is time-dependent, and EBV DNA in cells treated with L-I-OddU could rebound to pretreatment levels once the drug was removed. In view of the potent antiviral activity plus favorable toxicity profiles, L-I-OddU may be potentially useful for the treatment of EBV-related infectious diseases as well as for delaying the onset or decreasing the incidence of EBV-associated cancers.