Selenium (Se)-containing proteins in microsomal fractions of rat kidney and liver were investigated after isotopic labeling of rats with [75Se]selenite. More than 85% of the 75Se in the solubilized microsomal extracts precipitated with protein after trichloroacetic acid treatment. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), used to separate the labeled protein subunits in the solubilized microsomal extracts, revealed several 75Se-containing proteins in addition to glutathione peroxidase. 75Se-labeled subunits with molecular weights of 55, 30, 26, 22, 19, and 17 kDa were present in microsomal fractions of kidney and liver. The 75Se-labeled tryptic peptide of the 55 kDa subunit had the same Rf value on a 17% SDS-PAGE gel as the peptide from plasma selenoprotein P. A time-course study of the labeling of individual protein subunits in kidney and liver microsomes from Se-supplemented and Se-deficient rats showed that most of the 75Se was associated with the 55 kDa subunit 3 hr after injection. The amount of 75Se associated with this protein subunit decreased by 12 hr, with a concurrent increase in the labeling of lower molecular-weight subunits. The results support the hypothesis that there is a mechanism for transfer of Se from the 55 kDa subunit to other Se-containing proteins.