Twenty-two rabbits were given repeated subcutaneous injection of cadmium chloride. The cumulative cadmium dose given ranged from 13 to 214 mumole/kg body weight. Five rabbits served as controls. The treatment resulted in cadmium concentrations in kidney cortex that ranged from 0.3 to 3.2 mmole Cd/kg and a subsequent production of metallothionein. The molar ratio of cadmium, zinc, and copper in metallothionein fractions from kidneys with different concentrations of cadmium was determined. At low concentrations of cadmium in rabbit kidneys, zinc was the dominating metal bound to metallothionein (70-90%). At high concentrations of cadmium in kidneys, cadmium was the dominating metal in metallothionein. Evidence of kidney toxicity, in the form of beta2-microglobulinuria, was seen when cadmium constituted 85% of the metal ions recovered from metallothionein fractions. The remaining 15% was zinc. This indicates that at most six of the seven metal-binding sites in mammalian metallothionein are occupied by cadmium and that the remaining site is occupied by zinc. Our data provide further support for the hypothesis that chronic cadmium nephrotoxicity develops when there is a lack of metal-binding sites available for cadmium in metallothionein.