Patients on conventional hemodialysis have low levels of 25-hydroxy-vitamin D probably due to diet and decreased cutaneous synthesis. As 1,25 dihydroxy-vitamin D synthesis is substrate-dependent in end-stage renal disease, this could be a contributing factor to low 1,25 dihydroxy-vitamin D levels in patients undergoing conventional hemodialysis. We converted 35 patients historically on conventional hemodialysis to nocturnal hemodialysis for a minimum of 6 months thereby significantly increasing sessional equilibrated Kt/V from an average of 1.30 to an average of 2.01. Dietary restrictions were also removed. Serum phosphorus significantly fell, whereas the serum calcium, parathyroid hormone, and the mean dose of calcitriol did not change after the conversion. Significant increases in both 25-hydroxy and 1,25-dihydroxy-vitamin D levels were seen after hemodialysis mode conversion. A significant correlation was found between the dialysis dose and the levels of both hydroxylated forms of vitamin D. We suggest that improving uremia by nocturnal hemodialysis in the absence of exogenous supplementation is associated with increased 25 and 1,25-hydroxy-vitamin D levels. Additionally, normalization of serum phosphorus may improve 1alpha-hydroxylation thereby enhancing substrate-dependent generation of 1,25-dihydroxy-vitamin D in chronic dialysis patients.