A sensitive, precise assay for vitamin D in plasma is described. Three to five milliliters of plasma were extracted with methanol:methylene chloride (2:1). The lipid extract was chromatographed on Sephadex LH-20 and then on lipidex-5000 columns. After high pressure liquid chromatography with a reverse phase chromatographic system, vitamin D2 and vitamin D3 were quantitated by ultraviolet absorbance. We used this assay system for monitoring daily changes of vitamin D3 in plasma of two Jersey cows after four intramuscular doses (15 x 10(6) IU) of vitamin D3 administered at weekly intervals. Basal vitamin D in plasma was 3.2 +/- .99 ng/ml with a range of 1.7 to 4.9 ng/ml. Vitamin D3 in plasma remained relatively low (10 to 45 ng/ml) the week after the first vitamin D3 injection. Vitamin D3 was high (130 to 234 ng/ml) after the second, third, and fourth injections. Vitamin D3 decreased steadily to 88 ng/ml by 38 days after the fourth vitamin D3 injection. Phosphorus in plasma increased sharply to a plateau at 9.5 mg/100 ml during the week after the second vitamin D3 injection and returned to normal (4.5 mg/100 ml) at the end of the experiment. Calcium, however, gradually increased to 14.0 mg/100 ml 20 days after the fourth vitamin D3 injection. Both animals remained hypercalcemic (calcium 11.5 mg/100 ml) during the experiment.