Metastatic colonization of a secondary organ site is initiated by the attachment of blood-borne tumor cells to organ-specific adhesion molecules expressed on the surface of microvascular endothelial cells. Using digital video imaging microscopy and fluorescence activated cell sorting techniques, we show here that highly metastatic cells (B16-F10 murine melanoma and R3230AC-MET rat mammary adenocarcinoma cells) previously labeled with the fluorescent dye BCECF begin to transfer dye to endothelial cell monolayers shortly after adhesion is established. The extent of BCECF transfer to endothelial cell monolayers is dependent upon the number of BCECF-labeled tumor cells seeded onto the endothelial cell monolayer and the time of coculture of the two cell types, as visualized by an increase in the number of BCECF-positive cells among cells stained with an endothelial cell-specific mAb. Dye transfer to BAEC monolayers proceeds with a progressive loss of fluorescence intensity in the BCECF-labeled tumor cell population with time of coculture. The transfer of dye is bidirectional and sensitive to inhibition by 1-heptanol. In contrast, poorly metastatic B16-F0 melanoma cells and non-metastatic R3230AC-LR mammary adenocarcinoma cells do not efficiently couple with vascular endothelial cells. It is inferred from these experiments and from the amounts of connexin43 mRNA expressed by tumor cells that tumor cell/endothelial cell communication is mediated by gap junctional channels and that this interaction may play a critical role in tumor cell extravasation at secondary sites.