The level of serum 25-hydroxyvitamin D (25-OH-D3) was determined in 94 cases of healthy volunteers and 98 patients with various diseases. The mean level of the controls was 16.2 +/- 4.6 ng/ml (x /- S). The patients were divided into 5 groups, 1) Osteomalacia and rickets 42 cases, showing typical changes of bone in X-ray films. In 12 of them the disease was caused by vitamin D deficiency. The mean value of 25-OH-D3 was 3.3 +/- 2.0 ng/ml, being much lower than that of controls (P less than 0.001). In 14 cases of hypophosphatemia, 8 cases of renal tubular acidosis and 8 cases of renal insufficiency all complicated with osteomalacia the mean value of 25-OH-D3 was 17.9 +/- 11.4, 18.2 +/- 9.6 and 19.2 +/- 5.6 ng/ml (P greater than 0.05) respectively. 2) Intestinal malabsorption 18 cases. The mean level of 25-OH-D3 was 4.5 +/- 3.2 ng/ml (P less than 0.001). Some of them had hypocalcemia and/or secondary hyperparathyroidism. 2 showed osteoporosis and 1 osteomalacia. 3) Chronic liver disease 24 cases, the mean value of 25-OH-D3 was 6.2 +/- 5.6 ng/ml, being much lower than that of the controls (P less than 0.001). 4) 10 cases after taking anti-epileptic drugs had a mean level of 25-OH-C3 6.9 +/- 5.8 ng/ml (P less than 0.001). 1 had X-ray evidence of osteomalacia. 5) 4 cases of overdosage of vitamin D had a mean value of 110.3 +/- 22.5 ng/ml, being much higher than that of the controls (P less than 0.001). Competitive protein binding assay for measuring 25-OH-D3 is a simple and economic method. It is sensitive and specific as it provides distinct discrimination between healthy controls and patients with vitamin D deficiency or overdosage.