Monoclonal antibodies were prepared to localize the domain(s) of laminin to which tumor cells adhere. Rat Y3-Ag 1.2.3 myeloma cells were fused with spleen cells from a rat immunized with a purified 440-kDa fragment of chymotrypsin-digested laminin. Three monoclonal antibodies (AL-1 to AL-3) that bound to intact laminin in a solid-phase radioimmunoassay were chosen for further analysis. The epitopes recognized by these antibodies were characterized by radioimmunoassays, immunoblotting, radioimmunoprecipitation and immunoaffinity chromatography. In cell adhesion assays, monoclonal antibody AL-2 inhibited the binding of the highly metastatic melanoma cell line, K-1735-M4, to both intact laminin and the 440-kDa fragment of laminin. Electron microscopic examination of laminin-monoclonal antibody interactions showed that monoclonal antibody AL-2 reacted with the long arm of laminin directly below the cross-region. Two monoclonal antibodies that failed to inhibit tumor cell adhesion to laminin reacted with epitopes on the lateral short arms or cross-region of laminin as seen by electron microscopy. These results suggest that a new tumor cell binding domain of laminin may be located close to the cross-region on the long arm of laminin.