The subcellular distribution of selenium in rat tissues was studied by measuring 75Se in animals provided for 5 months with [75Se]selenite as the main dietary source of selenium. Equilibration of the animals to a constant specific activity allowed the measurement of 75Se to be used as a specific elemental assay for selenium. Of the whole-body selenium, 51% was in the soluble fractions and 48% was bound to the particulate fractions as follows: 21% in plasma membranes, 11% in microsomes, and 16% in mitochondria. Glutathione peroxidase was primarily a soluble enzyme, but part of the activity was associated with plasma membrane in liver, mitochondria in liver and kidney, and microsomes in testes. Selenium in glutathione peroxidase accounted for about one-third of the particulate-associated selenium. These results indicate that other selenium-containing proteins besides glutathione peroxidase are present in membranes.