The random cell migration of four human melanoma cell lines on laminin and type IV collagen-coated substrates was studied by video time-lapse image analysis and compared to the expression of a number of β₁ integrins including α₁β₁, α₂β₁, α₃β₁, and α₆β₁ using flow cytometry. These integrins were heterogeneously expressed in the four cell lines tested with three of four lines expressing α₂β₁. The melanoma cell line that did not express α₂β₁ exhibited weak attachment and low cell migration rate on both laminin and type IV collagen, whereas the other melanoma cell lines showed an increase in attachment and mean cell migration rate in a dose-dependent manner on the matrix molecules (p < 0.001). The enhanced migration seen in the three cell lines could be specifically inhibited by function blocking anti-β₁ and anti-α₂ monoclonal antibodies (p < 0.001) but not by function blocking anti-&alpha₃ and anti-α₆ monoclonal antibodies. Image analysis of the cells before and after treatment with anti-β₁ and anti-α₂ MoAb indicated that the inhibition of migration did not result in detectable cell detachment, retraction of cell processes, or other significant cell-shape change. Taken together, the findings suggest that the observable enhanced migration on laminin and type IV collagen of a number of human melanoma cell lines is largely mediated by integrin α₂β₁.