Background: Intercellular contacts between adjacent cells migrating over each other are important in many cellular processes. However it has been difficult to visualize and identify dynamic intercellular adhesions between migrating cells in situ. Methods: Two fluorescent membrane dyes, PKH2 and PKH26 for staining HT1080 and hematopoietic cells and cell lines, and an automated fluorescence microscopy system were used to monitor intercellular adhesion. Results: Cellular extensions connecting two or more adjacent cells were visualized, showing the intercellular adhesion between migrating cells for minutes and up to hours. After cells adhered to each other, followed by cell migration in different directions, cellular extensions were dragged from the pivotal contact points in different focal planes. CD34 -enriched mobilized peripheral blood cells and six hematopoietic cell lines showed intercellular connections in cocultures with HT1080. However, the frequency of intercellular connections was variable in different cocultures. A cell density of about 3.1 x 10 cells/cm for both cell lines in cocultures provided an adequate number of cells in each field of view, showing up to four intercellular connections per 100 total cells plated. Discussion: The tools derived from this study will open new areas of investigation for understanding the mechanism of the intercellular adhesion process. (C) 2000 Wiley-Liss, Inc.