The in vitro antiviral activity of a new series of cycloSal-pro-nucleotides derived from the acyclic nucleoside analogues aciclovir and penciclovir against herpes simplex virus type 1 (HSV-1), thymidine kinase deficient (TK-) HSV-1, and Epstein-Barr virus (EBV) was evaluated. Using the XTT-based tetrazolium reduction assay EZ4U, the cycloSal derivatives were examined for their antiviral and cytotoxic effects in HSV-1 as well as HSV-1-TK--infected Vero cells. The anti-EBV activity was assessed by means of an EBV DNA hybridization assay using a digoxigenin-labeled probe specific for the Bam H1-W-fragment of the EBV genome and by measuring viral capsid antigen (VCA) expression in P3HR-1 cells by indirect immunofluorescence. Among the new cycloSal-phosphotriesters the three aciclovir monophosphates proved to be potent and selective inhibitors of HSV-1 replication, EBV DNA synthesis and EB-VCA expression. Of interest is the retention of activity of the aciclovir monophosphates in HSV-1-TK--infected cells. Particularly 3-methyl-cycloSal-aciclovir monophosphate retained the same effectiveness, as compared to the wild type virus strain. In contrast to the aciclovir pro-nucleotides the penciclovir cycloSal-phosphotriesters exhibited at best only a marginal antiviral effect on HSV and EBV replication.