Abnormal gap junction communication (GJC) has been associated with carcinogenesis. We investigated the role of endothelial cell GJC and connexin 43 (Cx43), the main gap junction protein in these cells, during tumour cell extravasation. GJC was determined by the ability of cells to transfer Lucifer yellow, to neighbouring cells. Tumour-endothelial interaction was assessed by DiI assay. Connexin 43 expression and tyrosine phosphorylation were measured by immunoprecipitation and Western blotting. Co-culturing of ECV304 endothelial cells with human breast cancer cells resulted in a rapid and transient loss of communication competence of ECV304. This inhibition was maximal within 5 min. GJC was almost fully restored in 2 h. The co-culturing also resulted in an increase in the tyrosine phosphorylation of CX43. The pattern of phosphorylation was similar to the loss and the recovery of GJC in ECV304. We conclude that interaction of tumour cells with endothelium effectively inhibits GJC of endothelial cells, which is attributed to the increased tyrosine phosphorylation of connexin 43. This may contribute to the extravasation of tumour cells from the circulation, an essential step in the establishment of metastasis.