Immunofluorescence on primary dissociated rat neuronal cultures (cortical, hippocampal, and cerebellar) and organotypic hippocampal cultures was used to investigate the pattern of distribution of cell-surface amyloid protein (APP). Antibodies directed against the extracellular (N-terminal) portion of APP or against the entire molecule, but not against the C-terminal portion, revealed a striking segmental pattern of immunoreactivity along both axons and dendrites of all neuronal types tested. The pattern first developed between 24 and 48 h in culture. The segments showed co-localization with beta 1-integrin and talin immunoreactivities, but not with GAP-43 or clathrin, indicating that they may mark adhesion patches. Confocal laser microscopy supported a surface location for the APP responsible for the segmented pattern on neurites, as did the reduction of segmental immunoreactivity after exposure to mu-calpain or trypsin. It is conjectured that APP may have a role in cell-substratum interactions in the medium term, during such events as synaptic plasticity and neurite stability during extension.