Unlike iron therapy, folate use is not a standard of care in hemodialysis (HD) patients. Despite iron repletion, poor response to erythropoietin (EPO) treatment is common. Theoretical evidence for folate deficiency (FD) includes chronic blood loss, inflammation, malnutrition, and nutrient loss during dialysis. Due to poor diagnostic standards, early studies failed to establish a role for FD in EPO resistance. Given that hematological response to therapeutic intervention is the gold standard for FD, its diagnosis was therefore based on composite scoring of RBC and/or folate indices. Fifteen subjects (8-20 years) on chronic HD were enrolled in this study. No folate supplement was given in the first six months. Thereafter, 5-mg folic acid was administered orally after HD sessions over a six-month period. Folate indices before and after treatment were compared using percentage differences and paired t-tests. After folate use, the mean Hb increased by 11.4%, while MCV and RDW were reduced. Similarly, 4 of the 15 subjects each had a > or = 20% rise in Hb and a > or = 5% reduction in MCV, while 46.7% had a > or = 2.5% reduction in RDW. Mean RBC folate increased by 24%, while FD scores reduced from 3.8+/-1.2 to 0.4+/-0.7, and the EPO requirement by 90%. In contrast to previous studies, 26.7% of study subjects met the criteria for FD. Furthermore, the substantial (post-folate) reduction in the EPO requirement validates the need for therapeutic intervention, and therefore the presence of functional FD in the population.