Epithelial/epidermal fatty acid-binding protein (E-FABP) is induced in peripheral neurons during nerve regeneration and is found at high levels in central neurons during neuronal migration and development. Furthermore, E-FABP expression is required for normal neurite outgrowth in PC12 cells treated with nerve growth factor (NGF). The present study examined whether E-FABP plays a role in retinal ganglion cell (RGC) differentiation and axon growth. Rat retinal tissues from embryonic (E) and postnatal (P) development through adulthood were examined using immunocytochemical labeling with E-FABP and growth-associated protein 43 (GAP-43) antibodies. E-FABP colocalized with GAP-43 at E14 through P10. At E14, E-FABP immunoreactivity was confined to the somas of GAP-43-positive cells in the ganglion cell layer, but it was localized to their axons by E15. The axons in the optic nerve were GAP-43-positive and E-FABP-negative on E15, but the two proteins were colocalized by E18. Retinal cultures at E15 confirmed that E-FABP and GAP-43 colocalize in RGCs. Postnatally, labeling was present between P1 and P10 but decreased at older ages and was minimally present or absent in adult animals. Western immunoblotting revealed that at E18, P1, and P10 E-FABP levels were at least fourfold greater than those in the adult. By P15, protein levels were only twofold greater, with adult levels reached by P31. Furthermore, E-FABP could be reinduced during axon regeneration. Dissociated P15 retinal cells cultured in the presence of brain-derived neurotrophic factor, ciliary neurotrophic factor, and basic fibroblast growth factor exhibited sixfold more GAP-43 and E-FABP double-positive RGCs (cell body and axons) than controls. Moreover, all GAP-43-immunoreactive RGCs were also positive for E-FABP. Taken together, these results indicate the following: 1) E-FABP is expressed in RGCs as they reached the ganglion cell layer and 2) E-FABP plays a functional role in the elaboration of RGC axons in both development and regeneration.