Vitamin D (Vit D) is an essential element for the regulation of serum calcium, phosphate, and alkaline phosphatase (Alk Ph). Because the Vit D serum level is not usually measured directly, Vit D deficiency is diagnosed indirectly by changes in serum calcium, phosphate, and Alk Ph leves. The current study assessed the status of these biochemical parameters in subjects with different degrees of Vit D deficiency. We selected 1210 subjects, between 20 and 69 years old, randomly from the Tehran population. Subjects with diseases or medications that modified bone metabolism were excluded from the study. Serum 25(OH) D, calcium, phosphate, Alk Ph, and parathyroid hormone (PTH) levels were measured and the status of these biochemical parameters was compared in subjects with different degrees of Vit D deficiency. Vit D deficiency was diagnosed in 79.6% of the subjects. Different degrees of Vit D deficiency were classified as follows: group 1, severe; group 2, moderate; and group 3, mild. Serum PTH levels in the Vit D-deficient groups were significantly higher than that in group 4 (normal Vit D). Serum calcium and phosphate levels in groups 1 and 2 were significantly lower than those in groups 3 and 4. No significant difference was seen in serum Alk Ph in the groups with different degrees of Vit D deficiency. The sensivity for at least one biochemical variable (calcium, phosphorus, or Alk Ph) for the detection of severe, moderate, and mild Vit D deficiency was 24.2%, 13.8%, and 6%, respectively. When the serum 25(OH) D level was reduced to less than 25 nmol/l (groups 1 and 2), the effects of Vit D deficiency on calcium and phosphate levels were obvious. Therefore, the usual biochemical parameters (calcium, phosphate, Alk Ph) alone do not have sufficient sensitivity to detect mild deficiency of Vit D.