The cell adhesion, spreading and neurite-promoting properties of mouse tumor laminin fragment E8, which contains major site(s) responsible for laminin-cell interactions, were probed by proteolytic degradation, denaturation, synthetic peptides and antibody inhibition. Removal of more than half of the N-terminal portion contributing to the rod-like domain did not effect cell attachment or spreading although neurite-promoting activity was reduced. More extensive degradation of the rod or of the globular domains of E8, or separation of the globule from the rod, also resulted in loss of cell spreading activity although weak attachment was found to an A chain subfragment comprising the globular domain and a short piece of the rod. Exposure of E8 to increasing concentrations of dissociating agents produce an apparently reversible denaturation but an irreversible loss of both attachment and neurite-promoting activities, as did reduction and alkylation of disulfide bonds in the globular domain. Although cell adhesion and spreading were blocked by antibodies to an alpha 6 integrin subunit, neurite outgrowth was unaffected, indicating two distinct receptors for these two activities. Furthermore, a synthetic peptide, the sequence of which is found in the vicinity of adhesion and neurite-promoting sites and previously implicated in neurite growth and cell attachment activities, was found to be inactive. These results indicate that the major cell attachment and neurite-promoting sites of laminin are distinct although both require the native conformation of parts of the rod and the terminal globular domain of the long arm of laminin.