The aminoglycoside antibiotics have been the drug of choice for the treatment of tuberculo-sis and resistant cases of septicemia, but their use has been selective and restricted due to their known toxicities specially the ototoxicity and nephrotoxicity. Besides extensive litera-ture on the various side effects of aminoglycoside antibiotics in general and neomycin in particular, there is paucity of literature regarding the mechanism of toxicity. Although most of the studies highlight peripheral toxicities of neomycin, scientists have not sufficiently studied its central neurotoxicity.Taking these into considerations, the study was planned to know the effects of neomycin on the biochemical parameters (specially, sodium, potassium and calcium) in the central nervous system of albino rats. The present study was carried on 12 healthy, adult rats of either sex weighing 180±10 gm obtained from animal house of JNMC, AMU, Aligarh. Neomycin 100mg/kg body weight was given intramuscularly, every day for 10 days to the experimental animals and equal volume of distilled water, in identical manner to the control animals. The animals were decapitated on 10th day; brain and spinal cord were removed within 30 seconds and blotted on filter paper. The cerebrum, cerebel-lum, brain stem and spinal cord were separated and weighed to the nearest of milligram on a single pan electrical balance. Tissue samples were homogenized and digested in concen-trated nitric acid (100mg/ml). The supernatant solution was used for estimation of sodium and potassium by Flame Photometry and calcium level by the method of Clark and Collip and zinc and copper by the method of Donaldson and Pierre. Separate homogenate in dis-tilled water were centrifuged to get the supernatant for the estimation of total proteins in all the four parts by the method of Folin and Crocalteu. It was found that all the three cations i.e. sodium, potassium and calcium showed an increment in different regions of CNS and a zone of inhibition was observed after overnight incubation of CNS homogenate. Although there was a uniform response of different region of CNS for potassium concentration, the sodium was increased in the cerebrum, cerebellum and brain-stem while calcium was only increased in spinal cord. It was concluded that neomycin penetrates the central nervous tis-sue and central cause of muscular weakness after neomycin intoxication can’t be ruled out.