Live-imaging techniques (LIT), using specific dyes and quantifying the resulting fluorescence signals under a confocal microscope, have traditionally been used with isolated cells and tissues, to study various physiological parameters in-vitro. In the present study we report the successful application of LIT to complete gill filaments for the analysis and characterization of reactive oxygen species (ROS) and nitric oxide (NO) formation. Our results indicate that H2O2 (hydrogen peroxide), HOO• (peroxyl radical) and ONOO- (peroxynitrate anion) radicals (assessed through C-H2DFFDA staining) are mainly formed in the blood sinus of the filaments. Contrary, in the periphery of the filaments, with the lateral cilia and highest mitochondrial densities (MTK Deep Red 633 staining), staining with O2•- (DHE) and NO (DAF-2) sensitive dyes was highest. These peripheral areas also show the most acidic pH values (as observed with the dye ageladine-a) and mitochondria with low membrane potentials (JC-1), suggesting that the high amounts of ATP required for ciliary beating may be produced via a non-mitochondrial pathway such as the enzymatic activity of an ATP-regenerating kinase. On the ventral bend of the gills a high number of subepithelial vesicles were observed, which were especially numerous in the areas close to the mouth. These vesicles were brightly stained with NO and O2•- reactive dyes, while other ROS were absent. We hypothesize that these vesicles may be involved in mucus production in the gills and, given the antimicrobial properties of O2•-, release of this radical together with the mucus may be playing an important role in avoiding the development of microbial biofilms on the gill surface.