Autonomic blocked rats were given N^G-nitro-L-arginine methyl ester (L-NAME, 7.5 mg/kg) and showed a 18 % increased heart rate and a 48 % increased mean arterial pressure. Thyroidectomy, simultaneous to autonomic blockade, hampered the chronotropic response but did not modify the effect on blood pressure. After 150 min of autonomic blockade, the experimental end point, there was a 61 % decrease in total nitric oxide (NO) production by heart nitric oxide synthases (NOS), from 54 to 21 nmol NO/min.g heart. The activities of mtNOS and sarcoplasmic reticulum eNOS were 74 % and 52 % decreased, respectively. Mitochondria isolated from whole heart showed a well coupled oxidative phosphorylation with high respiratory control and ADP:O ratios, decreased mtNOS activity (55-60 %) and decreased mtNOS protein expression (70 %). Immunohistochemistry with anti-iNOS antibody linked to gold particles localized mtNOS at the inner mitochondrial membranes. Histochemical right atrium NOS (NADPH-diaphorase) was 55 % decreased after heart denervation. The effects of autonomic denervation on NO system were partially prevented by thyroidectomy performed simultaneously to autonomic blockade. Western blot analysis indicated a mtNOS very rapid protein turnover (t_1/2 = 120 min) with a process of protein expression that was up-regulated by thyroidectomy and with a degradation process that was down-regulated by the autonomic nervous system. The observations suggest that NO-mediated pathways contribute to pacemaker heart activity, likely through the NO steady-state levels in the right atrium and in the whole heart.