Nucleoside pools of herpes simplex type 1 (HSV-1)-infected and uninfected African green monkey kidney (GMK) cells and human fetal lung fibroblasts (HL) have been analysed with high-performance liquid chromatography (HPLC). The only nucleosides found in measurable amounts were deoxythymidine (dThd) and adenosine (Ado). The dThd pool seemed to be greater in GMK cells than in HL cells. dThd was also the only nucleoside excreted into the medium. HSV-1 infection reduced the dThd concentration of GMK cells. Addition of acyclovir (ACV) to HSV-1-infected GMK cells inhibited virus replication. This resulted in a dThd concentration similar to that of uninfected GMK cells. dThd added to HSV-1-infected GMK and HL cells reduced the antiviral action of ACV but not that of phosphonoformic acid (PFA). ACV is known to be activated mainly by HSV-induced deoxythymidine kinase (dTK), an enzyme which utilizes dThd as a substrate, while the action of PFA is independent of dTK. The low antiviral activity of ACV in GMK cells as compared to HL cells may be explained by the presence of high amounts of dThd in GMK cells.