Using newly developed and established extraction, Lipidex-5000 chromatography, normal phase gradient HPLC, and ligand binding assay techniques we have directly measured plasma and urine levels of vitamin D3 and its metabolites in seven normal subjects and seven patients with nephrotic syndrome and normal renal function. Significant reductions in the plasma levels of vitamin D3, 24,25(OH)2D3, 25,26(OH)2D3, and 1,25(OH)2D3 were noted in all nephrotic patients. In conjunction with the plasma metabolite abnormalities, direct quantitative analysis of the urine in these patients revealed significant increases in nonconjugated 250HD3, 24,25(OH)2D3 and 1,25(OH)2D3. Significant correlations were noted between the plasma and/or urine metabolites and other mineral homeostatic parameters. The results indicate that the primary basis for the reductions in plasma vitamin D3 and its metabolites in the nephrotic syndrome is enhanced urinary excretion. The findings of normal serum ionized Ca and i-PTH levels in the patients with nephrosis suggest that reductions in bound and not free forms of vitamin D3 metabolites in plasma may occur in the initial stages of the nephrotic syndrome.