Organic nitrates act as vasodilators and have long been used for treatment of cardiovascular diseases. However, the therapeutic effect of nitrates is limited by induction of nitrate tolerance which is associated with endothelial dysfunction and enhanced vasoconstriction. Multiple mechanisms cause nitrate tolerance including alterations in gene expression. To identify genes whose expression is altered due to chronic treatment with nitroglycerin (NTG), nitrate-tolerant rats were made by infusion of NTG for 3 days (10 µg/kg/min) using an osmotic minipump. We constructed a PCR-selected cDNA subtracted library from NTG-treated and vehicle-treated rat aortas. Screening of ∼500 clones in the subtracted library showed that four genes were regulated by NTG treatment. Specifically, mRNA levels of β-globin, tropoelastin, gelsolin and a small G protein were confirmed to be upregulated consistently by NTG treatment. These identified genes may play important roles in the development of nitrate tolerance and represent promising candidates to understand the mechanisms of nitrate tolerance and endothelial dysfunction in the vasculature.