Expression of the extracellular matrix molecule thrombospondin (TSP) was examined following retrobulbar crush injury of the goldfish and mouse optic nerve. TSP was present within the glia limitans and surrounding axon fascicles of the control normal goldfish optic nerve, but was absent from the normal mouse optic nerve. Following crush injury of the goldfish optic nerve, TSP expression increased dramatically along the path of regenerating axons and returned to near normal levels following axonal outgrowth. In contrast, during the unsuccessful attempt at regeneration following crush injury of the mouse optic nerve, TSP expression was present only in glial fibrillary acidic protein (GFAP)-negative, macrophage-rich regions distal to ganglion cell axons. These results indicate that TSP expression is increased in a temporal pattern along the path of regenerating goldfish optic nerve axons and therefore may be involved in successful central nervous system regeneration. The absence of TSP in the environment encountered by damaged mouse optic nerve axons may correlate with the lack of regeneration observed in the mouse optic nerve.