End-stage renal disease (ESRD) is the inability of the kidneys to remove waste products from the blood. The most important factors causing ESRD that require hemodialysis are diabetes and hypertension. There are limited numbers of studies to evaluate tetrahydrobiopterin pathway in these patients. The aim of the study was to evaluate tetrahydrobiopterin pathway by measuring its important components, biopterin to creatinine concentrations and dihydropteridine reductase activities in diabetes and hypertension patients treated with/without hemodialysis. The patients undergoing hemodialysis were classified as diabetic nephropathy (n = 21), hypertensive nephropathy (n = 20) and others (n = 30), while the controls consisted of healthy subjects (n = 21), diabetic subjects (n = 23) and hypertensive subjects (n = 22) without any renal disorder. It was found that urinary biopterin to creatinine concentrations significantly increased in kidney failure patients undergoing hemodialysis compared to the healthy control group (p < 0.05). Additionally, there were significant differences in urinary biopterin to creatinine concentrations between diabetes or hypertension patients and their hypertensive or diabetic control counterparts (both p < 0.05). Our results indicated an alteration in tetrahydrobiopterin pathway in ESRD, and in the presence of secondary pathologies such as diabetes and hypertension in the patients undergoing hemodialysis, more considerable changes are observed in the pathway. © 2013 by Walter de Gruyter Berlin Boston.