A Nafion(5 pre-coats/2 dip-coats)-modified Pt sensor developed for real-time neurochemical monitoring has now been characterised in vitro for the sensitive and selective detection of nitric oxide (NO). A potentiodynamic profile at bare Pt established +0.9 V (vs. SCE) to be the most appropriate applied potential for NO oxidation. The latter was confirmed using oxyhaemoglobin and N(2), both of which reduced the NO signal to baseline levels. Results indicated enhanced NO sensitivity at the Nafion(5/2) sensor (1.67 +/- 0.08 nA microM(-1)) compared to bare Pt (1.08 +/- 0.20 nA microM(-1)) and negligible interference from a wide range of endogenous electroactive interferents such as ascorbic acid, dopamine and its metabolites, NO(2)(-) and H(2)O(2). The response time of 33.7 +/- 2.7 s was found to improve (19.0 +/- 3.4 s) when the number of Nafion layers was reduced to 2/1 and an insulating outer layer of poly(o-phenylenediamine) added. When tested under physiological conditions of 37 degrees C the response time of the Nafion(5/2) sensor improved to 14.00 +/- 2.52 s. In addition, the NO response was not affected by physiological concentrations of O(2) despite the high reactivity of the two species for each other. The limit of detection (LOD) was estimated to be 5 nM while stability tests in lipid (phosphatidylethanolamine; PEA) and protein (bovine serum albumin; BSA) solutions (10%) found an initial ca. 38% drop in sensitivity in the first 24 h which remained constant thereafter. Preliminary in vivo experiments involving systemic administration of NO and L-arginine produced increases in the signals recorded at the Nafion(5/2) sensor implanted in the striatum of freely-moving rats, thus supporting reliable in vivo recording of NO.