We investigated whether dietary factors that are known to increase 1,25-(OH)2-cholecalciferol production can deplete plasma 25-OH-cholecalciferol. Plasma concentration of 25-OH-cholecalciferol, its metabolism in vivo and activities of renal mitochondrial 25-OH-cholecalciferol 1-hydroxylase (1-OHase) and 25-OH-cholecalciferol 24-hydroxylase (24-OHase) were measured in rats fed various amounts of calcium (Ca) and phosphorus (P). All diets contained 5 micrograms (200 IU) cholecalciferol per 100 g. For rats fed the "normal" diet (0.7% Ca and 1.2% P) the mean plasma 25-OH-cholecalciferol level was 11.0 +/- 0.8 nmol/L, and the mean 1-OHase activity was 30 +/- 5 fmol/(mg X min). All rats fed the low Ca (0.014%) diet had 1-OHase activities above 200 fmol/(mg X min) and undetectable plasma 25-OH-cholecalciferol levels (less than 2.5 nmol/L). The chi-square test interrelating plasma 25-OH-cholecalciferol and dietary Ca showed statistical significance (P less than 0.001). The high activity of 1-OHase that resulted from dietary Ca restriction increased utilization of 25-OH-cholecalciferol to the point of causing depletion of this metabolite in the circulation.