Introduction of the herpes simplex virus type 1 thymidine kinase gene into tumor cells, followed by the administration of the antiherpes nucleoside analogue ganciclovir has been demonstrated to be effective in eliminating solid tumors in animals. The success of this combination treatment largely depends on the bystander effect, i.e. the killing of nontransfected tumor cells by activated drug carried over from the nearby herpes thymidine kinase (tk) gene-transfected cells. We evaluated the in vitro bystander effect of several antiherpes purine and pyrimidine nucleoside analogues, using a colorimetric assay. All pyrimidine nucleoside analogues, including (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU), showed low, if any, bystander killing effect. In contrast, purine nucleoside analogues, such as ganciclovir, were endowed with a pronounced bystander killer effect. Lobucavir (Cyclobut-G), a ganciclovir analogue, displayed a two- to three-fold more pronounced bystander killer effect than ganciclovir, eliminating, at a concentration of 10 microM, 75% and 90% of a cell population that contained 5% and 10% tk gene-transfected cells, respectively. These findings were corroborated by autoradiographic analysis that showed that 2'-3H-BVDU metabolites formed in the herpes tk gene-transfected tumor cells were much less efficiently incorporated in the DNA of bystander cells than 8-3H-GCV. This indicates that, under the same experimental conditions, BVDU metabolites are less prone to pass the gap junctions than GCV metabolites.