B-50 (GAP-43) is a neural, membrane-associated protein that has been implicated in neurite outgrowth and guidance. Following stable transfection of Rat1 fibroblasts with B-50 cDNA we observed a dispersed distribution of B-50 immunoreactivity in flattened resting cells. In contrast, motile cells exhibited high concentrations of B-50 at the leading edge of ruffling membranes, coinciding with actin polymerization. Time-lapse studies on Rat1 fibroblasts transiently transfected with B-50/EGFP revealed that large vesicles originated from the ruffling membranes. These large vesicles (pinocytes) were found positive for Thy-1, a GPI-anchored protein, but negative for rab-5, an early endosome marker. In primary hippocampal neurons B-50 also colocalized completely with the raft marker Thy-1. Antibody-mediated cross-linking of Thy-1 in hippocampal neurons resulted in a redistribution of the intracellular protein B-50 to Thy-1-immunopositive membrane patches, whereas syntaxin was mainly excluded from the patches, showing that B-50 is associated with rafts. Academic Press.