AIM: To investigate the safety of tissue plasminogen activator (tPA) intra optic nerve injection in rabbits. METHODS: Group 1 and 2 (6 eyes in each group) received injection of tPA 25μg and 12.5μg in 0.1mL balanced saline solution (BSS). Group 3 (6 eyes) received injection of 0.1mL BSS. Six eyes in Group 4 as a normal control received no injection. The eyes were examined with slit lamp biomicroscope, indirect ophthalmoscope, visual evoked potentials (VEP) and electroretinography (ERG) at 1, 3, 7, 14 and 28 days after injection. RESULTS: No evidence of optic nerve or retinal toxicity or physical damage were revealed by ophthalmoscopy, VEP, and ERGs after the injection of tPA into the optic nerve. The means of the latency of the first peak of the VEP were 24.6±1.5, 24.1±1.9, 24.0±2.0 and 24.6±1.3mS respectively for the above specified groups (P =0.4112). The means of the amplitude of the first peak of the VEPs were 124±42, 145±41, 132±48 and 117±29μV respectively (P =0.0649). The means of the latency of a-waves were 6.0±0.4, 5.9±0.4, 5.9±0.5 and 5.8±0.3mS respectively (P =0.6279). The means of the amplitude of a-waves were 110±14, 112±15, 110±16 and 108?1μV respectively (P =0.7248). The means of the amplitude of b-waves were 151±12, 148±14, 144±16 and 141?0μV respectively (P =0.0957). CONCLUSION: Injection of tPA upto 25μg in 0.1mL into optic nerve is well tolerated.