Rat kidney selenium (Se)-containing proteins were studied by isotopic labeling with [75Se]selenite or [75Se]selenomethionine via three routes: oral, intraperitoneal injection, and incubation of kidney slices with the isotope. The two major Se-containing proteins in kidney were fractionated and partially characterized. 75Se elution profiles from Sephadex G-150 chromatography were similar for each labeling protocol, except for the profile obtained following incubation of slices with [75Se]selenomethionine. Of the two major 75Se-containing proteins, the one eluting at the void volume during Sephadex G-150 fractionation had a subunit of 23,000 Mr. The 75Se-labeled tryptic peptide from this protein and a 75Se-containing tryptic peptide from glutathione peroxidase had the same elution time from an HPLC column. A 75,000 Mr 75Se-containing protein had a 65,000 Mr subunit, and the 75Se-labeled tryptic peptide from this protein eluted from the HPLC column before that of glutathione peroxidase. Glutathione peroxidase is the most abundant kidney selenoprotein. Injection of animals with 75Se is the method of choice for isotopic labeling of rat kidney Se-containing proteins. Appropriate methods were developed that can be used in future studies of kidney Se-containing proteins.