The erythrocyte-free, isolated perfused rat liver was used to study the incorporation of selenium into glutathione peroxidase. Gel filtration and ion exchange chromatography of liver supernatant demonstrated 75Se incorporation into glutathione peroxidase. A 9-fold excess of unlabelled selenium as selenite or selenide very effectively reduced 75Se incorporation from L[ 75Se]-selenocystine, but a 100-fold excess of unlabelled selenium as selenocystine was relatively ineffective as compared to selenite or selenide in diluting 75Se incorporation from [ 75Se]selenite. These results indicate that selenide and selenite are more readily metabolized than is selenocysteine to the immediate selenium precursor used for glutathione peroxidase synthesis, and suggest a posttranslational modification at another amino acid residue, rather than direct incorporation of selenocysteine, as the mechanism for formation of the presumed selenocysteine moiety of the enzyme.