Tumor cell metastasis involves a complex series of interdependent events, including repeated invasion of basement membranes. Studies from several laboratories have implicated tumor cell adhesion and migration in response to laminin as a major contributing factor in tumor cell invasion. The current studies address the direct role of type IV collagen in promoting tumor cell adhesion, spreading, and migration. The observations of type IV collagen-mediated cellular behavior are contrasted with cellular behavior on type I collagen. The highly metastatic K1735 M4 melanoma cell line adhered, spread, and migrated in response to type IV collagen in a concentration-dependent manner. Functional assays using well-defined proteolytic fragments of type IV collagen demonstrated that melanoma cells interact with multiple domains of this protein. Highly metastatic melanoma cells adhered, spread, and exhibited motile behavior in response to 0.2 to 200 nm concentrations of a purified pepsin-generated, triple helix-rich domain of type IV collagen. In contrast, cells adhered and spread but were essentially nonmotile in response to a purified major noncollagenous domain of the protein. In addition, de novo protein synthesis was required for cell adhesion to the major noncollagenous domain, whereas adhesion to the helical domain was less dependent upon de novo protein synthesis. Arg-Gly-Asp (RGD)-related peptides were used to study the adhesion and spreading of melanoma cells on type IV collagen. The results demonstrated that a serine containing RGD-related peptide (GRGDSP) has virtually no effect on melanoma cell adhesion on type IV collagen-coated substrata, whereas this peptide inhibited melanoma cell adhesion to fibronectin-coated substrata in a concentration-dependent manner. In contrast, when threonine was substituted for serine (GRGDTP), cell adhesion to type IV collagen was significantly (45%) inhibited. The threonine-containing peptide virtually eliminated cell adhesion on substrata coated with type I collagen. These data demonstrate that adhesion, spreading, and migration of melanoma cells on type IV collagen have a complex molecular basis which is partially dependent on RGD-related sequences.