Patients with the nephrotic syndrome may exhibit low serum 25-OH-D concentrations. We developed a method for isolation of 25-OH-D from urine, with measurement by competitive binding assay. Daily urinary 25-OH-D excretion in healthy subjects averaged 0.17 +/- 0.15 nmol/day. Among nephrotic patients, urinary 25-OH-D excretion ranged from 0.27 to 10 nmol/day, in direct relation to the severity of proteinuria (r=0.76) and averaged 3.7 +/- 3.5 nmol/day. The 25-OH-D in the urine of nephrotic patients was unconjugated, implying that it was excreted with the serum VDBG, which has been shown to have a molecular weight and isoelectric point similar to that of albumin. We conclude that low serum 25-OH-D concentrations among nephrotic patients are principally the result or urinary losses of steroid. (J Lab Clin Med 99:325, 1982.)