Patients on extended hours (>15 h/week) hemodialysis may be at a higher risk of deficiency of water-soluble vitamins than conventional (≤15 h/week) hemodialysis patients due to their increased weekly hours of dialysis. We compared serum levels of the water-soluble vitamins in a group of extended and conventional hours hemodialysis patients. Predialysis serum levels of vitamin C, vitamin B12, thiamine, pyridoxine, and folate were measured in 52 patients: 26 extended group and 26 conventional group. Information on patient's intake of vitamin supplements and dialysis regimen was obtained. Data were log transformed due to the skewed distribution of the results. Median vitamin C levels were significantly lower in the extended group (0.30 vs. 1.14 mg/dL, P<0.001), with 7 patients having a level <0.18 mg/dL. Thiamine levels were also lower in the extended group (median 211 vs. 438.5 nmol/L, P=0.0005). However, extended patients had higher levels of pyridoxine (23.2 vs. 11.1 ng/mL, P=0.03). Vitamin B12 and folate levels were not significantly different between the groups. There was significant variability in vitamin supplement prescription in both groups, and dietary data were not obtained. This study showed a high incidence of vitamin C deficiency in extended hours hemodialysis patients, suggesting that supplementation is warranted. It also supports an ongoing role for multivitamin supplementation in conventional hemodialysis patients.