The phosphorylation of thymidine and iododeoxyuridine by thymidine kinase was stimulated by 5-iodo-5'-amino-2',5'-dideoxyuridine ( AIdUrd ). Antagonism of the feedback inhibition that is normally exerted by the 5'-triphosphates of thymidine and iododeoxyuridine appears to account for the stimulation. The effect of AIdUrd on thymidine kinase purified from HeLa cells by affinity column chromatography was critically dependent on the presence of these feedback inhibitors. In the presence of thymidine triphosphate or iododeoxyuridine triphosphate, AIdUrd could markedly stimulate ( deinhibit ) enzyme activity, whereas, in their absence, AIdUrd inhibited thymidine kinase with an apparent Ki of 0.7 microM. Stimulation was evident over a wide range of concentrations of both iododeoxyuridine and adenosine triphosphate. In intact HeLa and Vero cells, phosphorylation of thymidine and iodoeoxyuridine was strongly enhanced by AIdUrd . Large increases in the intracellular levels of nucleotides derived from exogenous thymidine and iododeoxyuridine were apparent. As a consequence, the cytotoxicity of both nucleosides was exacerbated by AIdUrd . The reductions in cellular replication rates and colony formation produced by iododeoxyuridine were enhanced by AIdUrd . Although the replication of HeLa cells was not inhibited by either thymidine (30 microM) or AIdUrd (300 microM), in combination they were strongly synergistic and produced a 60% inhibition of cellular growth. Under these conditions, the uptake of thymidine was increased over 300% by AIdUrd . AIdUrd represents a new regulatory antagonist of thymidine kinase which may be useful in novel chemotherapeutic strategies.