GAP-43 is a palmitoylated protein in Cys residues at positions 3 and 4 that mostly localizes in plasma membrane (PM). We have examined membrane association, subcellular distribution and intracellular trafficking of GAP-43 in CHO-K1 cells. Using biochemical assays and confocal and video microscopy in living cells we demonstrate that GAP-43, at steady state, localizes at the recycling endosome in addition to the cytoplasmic leaflet of the PM and Golgi complex. Single or double mutation of Cys-3 or 4 of GAP-43 completely disrupts PM and recycling endosome association. A combination of selective photobleaching techniques and time-lapse fluorescence microscopy reveals a dynamic association of GAP-43 with recycling endosomes in equilibrium with the PM pool. Newly synthesized GAP-43 is found mainly associated with the Golgi complex, but not with recycling endosome, and traffics to PM by a BFA-insensitive pathway. Impairment of PM fusion and internalization by treatment with tannic acid does affect the trafficking of GAP-43 from PM to recycling endosomes which reveals a vesicles-mediated retrograde trafficking of GAP-43. Taken together, these results suggest that the recycling endosome-associated pool of GAP-43 represents a post biosynthetic fraction in equilibrium with the PM-associated GAP-43 and support new roles for GAP-43 in membranes from the pericentriolar endosomal compartment.