For the further investigation of nephrocalcinosis (NC) observed by us in male and female Sprague-Dawley rats fed purified diets (1), three experiments were conducted. In the first experiment the effect of the level of dietary protein on NC was investigated. Twenty male and 20 female weanling Sprague-Dawley rats were randomly divided (each sex separately) into 2 groups One group of each sex was fed, for 6 weeks, a purified diet containing 10% protein from casein and the other a diet containing 15% protein from the same source. The diets contained the AIN-76A vitamin and mineral mixtures. At the end of the experiment the kidneys of the rats in the 10% protein group contained (mg Ca/g dry weight, mean, minimum, maximum): male, 15.6, 0.3, 69.9; female, 49.0, 30.2, 69.1. The corresponding values for the animals in the 15% group were: male, 1.3, 0.2, 7.2; female, 24.0, 8.2, 48.4. Thus, the increase in the protein content of the diet decreased the kidney calcium levels. In the second experiment, three diets containing 20% protein from casein and 25 mg fluoride/kg with increasing levels of magnesium (0.10, 0.20, 0.27%) were tested in groups (10 each) of male and female weanling rats fed for 6 weeks. At the end, the calcium contents of the kidneys, expressed as above, were, for the diet with 0.10% Mg — male, 0.33, 0.29, 0.36; female, 0.98, 0.26, 6.22; and for the diet with 0.20% Mg — male, 0.30, 0.27, 0.34; female, 0.57, 0.29, 1.20; and for the diet with 0.27% Mg — male, 0.31, 0.29, 0.37; female, 0.54, 0.27, 1.07. These results indicated that a supplement of 25 mg fluoride/kg and a total magnesium content of 0.10% was sufficient to prevent NC in male rats but females fed the same level of fluoride and even 0.27% magnesium had kidney calcium levels somewhat higher than normal. The calcium, phosphorus and magnesium concentrations of the hearts in these animals were not affected. In the third experiment soy assay protein (SAP) was used at a 20% protein level with and without a supplement of 50 mg fluoride/kg and 0.22% magnesium (total 0.27%). Kidney minerals, urinary levels of minerals, creatinine and citrate were determined. Kidney calcium levels were somewhat lower than comparable casein-fed rats and the supplement prevented NC. The composition of urine indicated that females excreted more sulphate and calcium per g creatinine than males. This could explain the more severe NC found in female rats. The supplement caused an increase in the excretion of magnesium and citrate and a decrease in the urinary phosphorus level. These changes prevented NC in both male and female rats.