Enzymes involved in the metabolism nitric oxide (NO) and reactive oxygen species (ROS) may play a role for the decreased availability of NO in atherosclerosis. We, therefore, hypothesized that the pattern of gene expression of these enzymes is altered in atherosclerosis. Myocardial tissue from patients with coronary heart disease (CHD) or without CHD (control group) was investigated. The level of enzymes related to NO/ROS metabolism was determined both at mRNA level and protein level by rt-PCR, real-time PCR, and western blot. The expression of NOS1–3 (synthesis of NO), arginase1 (reduction of l-arginine), p22phox (active subunit of NADPH oxidase), GTPCH (rate limiting enzyme for tetrahydrobiopterin), SOD1–3 (scavengers of superoxide anions), PRTMT1–3, and DDAH2 (involved in the metabolism of ADMA) was determined. All enzymes were found to be expressed in human myocardium. NOS isoforms were decreased in CHD in protein level, but only the downregulation of NOS3 expression reached statistical significance. The expression of PRMT1 and PRMT3 was increased. In addition, the expression of DDAH2 was reduced, both theoretically leading to an increase of ADMA concentration. SOD3 was downregulated in tissue from patients with CHD. Taken together, in myocardial tissue from patients with atherosclerosis, the expression of genes increasing ADMA levels is enhanced in contrast to a reduced expression of genes promoting NO synthesis. These results may contribute to the explanation of increased oxidative stress in atherosclerosis on the level of gene expression.