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The buckminsterfullerene (C60) is considered as a relevant candidate for drug and gene delivery to the brain, once it has the ability to cross the blood-brain barrier. However, the biological implications of this nanomaterial are not fully understood, and its safety for intracerebral delivery is still debatable. In this study, we investigated if C60 particle size could alter its biological effects. For this, two aqueous C60 suspensions were used with maximum particle size up to 200 nm and 450 nm. The suspensions were injected in the hippocampus, the main brain structure involved in memory processing and spatial localization. In order to assess spatial learning, male Wistar rats were tested in Morris water maze, and the hippocampal BDNF protein levels and gene expression were analyzed. Animals treated with C60 up to 450 nm demonstrated impaired spatial memory with a significant decrease in BDNF protein levels and gene expression. However, an enhanced antioxidant capacity was observed in both C60 treatments. A decrease in reactive oxygen species levels was observed in the treatments with suspensions containing particles measuring with up to 450 nm. Thiobarbituric acid reactive substances, glutamate cysteine ligase, and glutathione levels showed no alterations among the different treatments. In conclusion, different particle sizes of the same nanomaterial can lead to different behavioral outcomes and biochemical parameters in brain tissue.Different particle sizes of C60 exhibited different behavioral effects.C60 (450 nm) treatment impaired spatial memory and decreased BDNF levels.Enhanced antioxidant capacity was observed in both C60 treatments (450 and 200 nm).Decrease in ROS levels was observed in the treatment with 450 nm sized particles.