In rats, phosphorus deficiency (P-) has been shown previously to stimulate the linear bone apposition rate (BAR) and this P- effect is dependent on adequate intake of vitamin D. To investigate further the relative importance of the vitamin D3 metabolites, 1,25(OH)2D3, 24,25(OH)2D3, and 25(OH)D3, in BAR stimulation, we studied, in P- rats, the relationships between BAR and plasma levels of these three vitamin D3 metabolites following vitamin D3 deprivation. Three groups of rats were placed on diets differing only in phosphorus (P) and vitamin D3(D3) content, with one group diet deficient in both P and D3, one diet, P-, D3 replete, and one diet both P and D3 replete. Plasma levels of the three vitamin D3 metabolites, plasma Ca and P, isotopic Ca absorption and BAR measurements were carried out at 1, 3, and 5 weeks after onset of the test diets. In P-, D3 replete rats, both plasma levels of 1,25(OH)2D3 and BAR were increased throughout the 1 to 5 week study period, while 25(OH)D3 and 24,25(OH)2D3 levels were not significantly different from P and D3 replete controls. In P-, D3 restricted rats, BAR was decreased by one week, prior to any reduction in plasma levels of 25(OH)D3 and 24,25(OH)2D3 and while plasma 1,25(OH)2D3 levels were still well above control values. In this P- rat model, the vitamin D dependent BAR stimulation does not appear to be directly related to alterations in the plasma levels of 1,25(OH)2D3, 24,25(OH)2D3, or 25(OH)D3.