The effects of pinealectomy and of intraperitoneally administered melatonin on the retrograde degeneration of retinal ganglion cells (RGCs) were examined in a novel model of optic nerve (ON) transection in C57BL/6J mice. RGCs were prelabeled with the fluorescent tracer 1,1′-dioctadecyl-3,3,3′,3′-tetramethyl indocarbocyanine perchlorate (Di-I), and the ON was cut inside the orbital cavity 7 days later. The degree of RGC injury was assessed by counting viable Di-I labeled RGCs in various locations of the retina. In unlesioned control eyes, a mean ganglion cell density of 1891 ± 30/mm2 (mean ± S.E.M.) was determined. The cell density markedly declined at 14 days after axotomy (295 ± 9 cells/mm2; 15.6% of contralateral). Sham-pinealectomy did not influence the density of RGCs at 14 days after ON transection (382 ± 37 cells/mm2). In pinealectomized animals, on the other hand, the RGC number was significantly reduced as compared with untreated and sham-pinealectomized animals (91 ± 33 RGCs/mm2). The effect of pinealectomy was reversed after i.p. administration of melatonin (4 mg/kg bw bolus followed by continuous infusion of 8 mg/kg bw/day) (286 ± 27 cells/mm2). In nonpinealectomized animals, on the contrary, i.p. melatonin did not influence the RGC density (344 ± 20 cells/mm2). The present results suggest that endogenous melatonin prevents the delayed degeneration of adult central nervous system (CNS) neurons in vivo, and that exogenous substitution of melatonin may be useful to protect injured neurons against cell death under conditions of melatonin deficiency, e.g. in the aged brain, when melatonin synthesis and secretion have decreased.