We describe a new, rapid, and simple column-chromatographic procedure for 25-OH vitamin D3 in plasma. The vitamin is extracted by use of Sep-pak C18 (octadecyl alkylated silicic acid), a short factory-packed reversed-phase column, and 25-OH vitamin D2 + 25-OH vitamin D3 fraction is eluted with methanol/water. The 25-OH D2 and 25-OH D3 are then well resolved on a high-resolution 3-micrometer silicic acid straight-phase liquid-chromatography column. The peaks are quantified against a 25-OH D3 standard by ultraviolet absorbance. Recovery was assessed by use of tritiated 25-OH D3. The within-assay coefficient of variation of the method was 5% and recovery 93%. The method was evaluated with 26 samples from control subjects and 17 samples from patients, seven with liver disease and 10 who had undergone ileo-ileostomy for hypercholesterolemia. The normal seasonal variation was observed for 25-OH D3 concentrations, and they correlated negatively and significantly with those of 25-OH D2. Post-ileo-ileostomy concentrations of 25-OH D3 in plasma were generally similar to those in normal individuals for the same reason (winter), but 25-OH D2 concentrations were insignificantly lower. The patients with chronic liver disease had significantly lower 25-OH D3 concentrations than normal persons but higher 25-OH D2 concentrations, with a significantly higher 25-OH D2/25-OH D3 ratio, indicating poor storage of vitamin D3.