A novel gap junction-independent mechanism for ganciclovir-mediated bystander effect killing by a herpes simplex virus thymidine kinase (HSV-TK)-expressing SW620 human colon tumor cell line has been characterized. The mechanism of the HSV-TK/GCV bystander effect for many tumor cell lines has been demonstrated to be due to connexin gap junction transfer of phosphorylated ganciclovir (GCV) metabolites; however, there may be as yet uncharacterized connexin-independent mechanisms for the effect. To address this, the bystander effect was further evaluated in a panel of cell lines mixed with homologous HSV-TK-expressing cell lines, a SW620.TK cell line, or a high connexin43-expressing PA-317.TK cell line. Of the 10 cell lines tested, 4 were found to be resistant to bystander effect killing by their homologous HSV-TK-expressing cell lines and the PA-317.TK cells, but all of the cell lines were sensitive to GCV killing when mixed with the SW620.TK cells. The SW620.TK cells were then further evaluated for any indication of extracellular GCV metabolite efflux. Culture medium from SW620.TK cells labeled with [(3)H]GCV was evaluated for the presence of GCV nucleotides by ion-exchange column separation and HPLC analysis. The presence of GCV mono-, di-, and triphosphate metabolites in the medium was detected. Inclusion in the medium of inhibitors of extracellular phosphatases and ecto-ATPases increased the proportion of GCV metabolites recovered. These results indicate that phosphorylated GCV metabolites can be effluxed from SW620.TK cells and that some type of cellular uptake mechanism independent of gap junctions exists for nucleotide entry into neighboring cells.