This is a prospective, controlled, randomized clinical study to evidence the role of nitrosative stress in development of overt neurological sign of canine distemper (CD). The enrollment of cases wasa made on basis of overt sign of CD (n=139) and the findings were compared with apparently healthy dogs (n=15). The CD specific immunoglobulins resulted in 94 confirmed positive cases. The nitric oxide (NO) and neuronal nitric oxide synthase (nNOS) concentration in cerebrospinal fluid (CSF) (18. 08 ± 2. 76, 415. 84 ± 46. 24, respectively) and plasma (32. 68 ± 4. 31, 321. 31 ± 102. 30, respectively) were significantly (P<0. 05) elevated as compared to healthy control group. The concentration of neuron specific enolase (NSE) in CSF and plasma were also significantly (P<0. 05) higher in dogs suffering from CD. The significant differences in other biochemical parameters like total protein, albumin and globulin were found in the CSF of dogs compared with healthy control. The author concludes that NO plays a role in pathophysiology of neurological form of CD and nNOS and NSE estimation in CSF and plasma could help in making early diagnosis of clinical cases