A reliable method of directly measuring endogenously generated nitric oxide (NO) in real-time and in various brain regions is presented. An extensive characterisation of a previously described amperometric sensor has been carried out in the prefrontal cortex and nucleus accumbens of freely moving rats. Systemic administration of saline caused a transient increase in signal from baseline levels in both the prefrontal cortex (13 ± 3 pA, n = 17) and nucleus accumbens (12 ± 3 pA, n = 8). NO levels in the prefrontal cortex were significantly increased by 43 ± 9 pA (n = 9) following administration of l-arginine. A similar trend was observed in the nucleus accumbens, where an increase of 44 ± 9 pA (n = 8) was observed when compared against baseline levels. Systemic injections of the non-selective NOS inhibitor l-NAME produced a significant decrease in current recorded in the prefrontal cortex (24 ± 6 pA, n = 5) and nucleus accumbens (17 ± 3 pA, n = 6). Finally it was necessary to validate the sensors functionality in vivo by investigating the effect of the interferent ascorbate on the oxidation current. The current showed no variation in both regions over the selected time interval of 60 min, indicating no deterioration of the polymer membrane. A detailed comparison identified significantly greater affects of administrations on NO sensors implanted in the striatum than those inserted in the prefrontal cortex and the nucleus accumbens.