In a previous work we showed that a decrease in the free intracellular magnesium produced alterations in the kinetic behaviour of 1 alpha-hydroxylase which reduced the synthesis rate of 1,25(OH)2D3. This in vitro result strongly supports that magnesium deficiency could also induce in vivo failure of the renal enzyme and then a decrease of 1,25(OH)2D3 serum levels. In the present work we have tested the effect of magnesium deficiency on the in vivo transformation of the substrate 3H-25(OH)D3 to 3H-1,25(OH)2D3 as well as the distribution of synthesized hormone among its different target tissues. We found that magnesium deficiency produced a decrease of both the in vivo synthesis of 3H-1,25(OH)2D3 and the binding of the radioactive hormone to bone tissue. These results may explain the different criteria present in the scientific literature concerning the relationships between magnesium status and vitamin D metabolism.