We identified a -786T>C polymorphism in the eNOS gene, and this polymorphism was strongly associated with coronary spasm. The present study aimed to elucidate whether the -786T>C polymorphism or acetylcholine (ACh)-induced coronary spasm affects serum nitrite/nitrate (NOx) levels. The study population comprised three groups: (i) 26 patients without coronary spasm in the left anterior descending coronary artery (LAD) with the T/T genotype (group A); (ii) 20 patients with coronary spasm in the LAD with the T/T genotype (group B); and (iii) 16 patients with coronary spasm in the LAD with the C/T genotype (group C). Paired blood samples were obtained from the coronary sinus (CS) and the aortic tract (Ao) before and after an intracoronary injection of ACh. Serum NOx and plasma lactate levels were measured. The delta NOx level was calculated as the serum concentration of NOx in the CS minus that in the Ao. We compared lactate extraction ratios (LERs) and delta NOx levels between the three groups. The LERs after the provocation test in groups A, B and C were 18.9+/-2.4%, -0.5+/-3.9% and -13.5+/-4.2%, respectively. The LER in group C was significantly lower than in group B. The delta NOx levels after the provocation test in groups A, B and C were 11.5+/-1.7 micromol/l, 10.4+/-3.5 micromol/l and -2.1+/-4.8 micromol/l, respectively. The delta NOx levels in group C were significantly lower (P<0.05). Although the NOx level was significantly increased after the provocation test in group A (P<0.05), the NOx level was significantly decreased after the provocation test in group C (P=0.001). In group B, the provocation test did not significantly change the delta NOx level. In conclusion, the -786T>C polymorphism reduces the NOx level from the heart due to an intracoronary injection of ACh, and thereby predisposes the patients to severe coronary spasm.