The influence of the thymidine (Thd) kinase (TK) of herpes simplex virus type 1 (HSV-1) on the intracellular uptake and anabolism of nucleosides has been investigated. To compare the differences between the TK-positive (TK(+)) and TK-deficient strains, acyclovir (ACV)-resistant strains were cloned from a cell culture and classified into 2 groups, viz. the TK-partial (TK(p)) and TK-negative (TK(-)). The cellular uptake of thymidine was highly dependent on the viral TK (vTK) activity. The TK(+) strain showed the highest level of intracellular thymidine uptake, the TK(p) strain a moderate level, which varied from strain to strain, and the TK(-) and mock strains showed little uptake. The inhibition of viral replication by ACV, ganciclovir (GCV) and penciclovir (PCV) did not decrease the Thd uptake at all. On the contrary, a notable increase found to be induced by ACV. The influence of the vTK on the uptake of GCV or PCV was much greater than that of ACV. The metabolism was generally less dependent on the vTK activity than the influx. The influx and phosphorylation rates of GCV and PCV were dependent on the substrate specificity of the vTK.