This study shows the presence of all three nitric oxide synthases (NOSs) and NOS activity in H9c2 cells cultured under non-stimulated conditions. By using the 4,5 diaminofluoresceindiacetate (DAF-2DA) fluorimetric nitric oxide (NO.) detection system we observed NO. production in H9c2 cells. As revealed by confocal microscopy, NO. fluorescence colocalizes in mitochondria labeled with Mito-Tracker Red CM-H(2)Xros. Upon stimulation with acetylcholine (Ach), which increased NOS activity by 75%, the colocalization coefficient C-green value, calculated as Pearson's correlation, increased from 0.07 to 0.10, demonstrating an augmented presence of NO. Mi mitochondria. Conversely, the presence of NO. in mitochondria decreased following cells pretreatment with L-MonoMethylArginine (L-NMMA), a competitive inhibitor of NOS activity, as indicated by the reduction of the C-green value to 0.02. This work confirms that the presence of NO. in mitochondria. can be modulated in response to different fluxes of NO. (C) 2002 Elsevier Science (USA).