Using unselected and selected B16 melanoma cell lines, we examined the relationship between homotypic aggregation properties and the potential to form experimental metastatic lung colonies. The B16 sublines were selected in vitro from a line with relatively low homotypic aggregation kinetics and experimental metastatic potential (B16-F1) by successive steps of cell aggregation, followed by separation of cell aggregates from single cells. The selected sublines possessed significantly higher rates of aggregation than did the parental cell line and, when injected intravenously as single cells, formed greater numbers of lung tumor colonies. The aggregation kinetics of the parental and selected cell lines were dependent on divalent cations, with the following order of selectivity: Ca2+ greater than Mn2+ much greater than Mg2+. Syngeneic and xenogeneic serum components and the protease inhibitor leupeptin enhanced the aggregation kinetics of various B16 cell lines. The results support the proposal that a positive correlation exists between increased homotypic adhesion and experimental metastatic potential.