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The use of isotope dilution analysis (IDA) with inductively coupled plasma–mass spectrometry (ICP–MS) for the determination of oxidized metallothionein (MT) by a Cd-saturation method is investigated. The method developed here is a modification of an earlier methodology which used a radioactive Cd isotope (¹⁰⁹Cd). While retaining the many advantages of this previous approach, the procedure presented here uses stable isotope ratio measurements (¹¹⁴Cd/¹¹¹Cd) for the determination of MT. Experimental parameters governing the instrumental precision and accuracy for isotope ratio measurements of Cd by ICP–MS were characterized. Systematic errors, including mass bias, detector dead time, and spectroscopic interferences, could be easily corrected. The isotope dilution ICP–MS method was validated by the determination of very low levels of cadmium in biological certified reference materials (NIST SRM 2670 freeze-dried urine, IAEA H-8 horse kidney, and BCR TP-25 lichens). Finally, the IDA procedure was evaluated for the determination of oxidized MT by a Cd-saturation method previously developed using radioactive ¹⁰⁹Cd. The final procedure was applied to the quantification of MT in Long–Evans Cinnamon rat liver cytosol samples and the results were compared with data obtained for the same samples using the reference ¹⁰⁹Cd methodology. A good agreement between the analytical values obtained by both methods was observed. Copyright 2000 Academic Press.