Phagokinetic assay demonstrated that B16-F1 melanoma cells cultivated in vitro as spheroids on a non-adhesive substrate produce more autocrine motility factor (s) as compared to cells cultured as a monolayer. Cells grown as spheroids showed more motile activity stimulated by conditioned media than cells grown as a monolayer. Fluorescence activated cell sorter analysis showed that cells cultivated as spheroids are less stained with the 3F3A monoclonal antibody directed against a Mr 78, 000 glycoprotein, which is identified as a membrane receptor for autocrine motility factor, compared to cells grown as a monolayer. These results suggest that enhancement of the production of autocrine motility factor (s) and regulation of its receptor endocytosis play an important role in the enhanced motility of cells grown as spheroids which enhance their ability to colonize the lung.