Hydroxyurea has been shown to potentiate the anti-human immunodeficiency virus activities of 2',3'-dideoxynucleoside analogs such as didanosine. We have now evaluated in vitro the effect of hydroxyurea on the antiherpesvirus activities of several nucleoside analogs (acyclovir [ACV], ganciclovir [GCV], penciclovir [PCV], lobucavir [LBV], (R)-9-[4-hydroxy-2-(hydroxymethyl)butyl]guanine [H2G], and brivudin and nucleoside phosphonate analogs (cidofovir [CDV] and adefovir [ADV]). When evaluated in cytopathic effect (CPE) reduction assays, hydroxyurea by itself had little effect on CPE progression and potentiated in a subsynergistic (herpes simplex virus type 1 [HSV-1]) to synergistic (HSV-2) fashion the antiviral activities of ACV, GCV, PCV, LBV, H2G, ADV, and CDV. Hydroxyurea also caused marked increases in the activities of ACV, GCV, PCV, LBV, and H2G (compounds that depend for their activation on a virus-encoded thymidine kinase [TK]) against TK-deficient (TK) HSV-1. In fact, in combination with hydroxyurea the 50% effective concentrations of these compounds for inhibition of TK HSV-1-induced CPE decreased from values of 20 to 100 µg/ml (in the absence of hydroxyurea) to values of 1 to 5 µg/ml (in the presence of hydroxyurea at 25 to 100 µg/ml). When evaluated in a single-cycle virus yield reduction assay, hydroxyurea at a concentration of 100 µg/ml inhibited progeny virus production by 60 to 90% but had little effect on virus yield at a concentration of 25 µg/ml. Under these assay conditions hydroxyurea still elicited a marked potentiating effect on the antiherpesvirus activities of GCV and CDV, but this effect was less pronounced than that in the CPE reduction assay. It is conceivable that the potentiating effect of hydroxyurea stems from a depletion of the intracellular deoxynucleoside triphosphate pools, thus favoring the triphosphates of the nucleoside analogues (or the diphosphates of the nucleoside phosphonate analogues) in their competition with the natural nucleotides at the viral DNA polymerase level. The possible clinical implications of these findings are discussed.