The distribution of selenium-binding proteins in specific tissues of mice and rats was examined by electrophoretic and immunologic techniques. Major selenium-binding proteins of 58K and 26K daltons were present in whole blood, erythrocytes, liver, duodenum, mammary tumors, kidney, testis, ovary and pancreas by electrophoretic analysis. By Western immunoblot it was evident that the 58K protein in plasma did not cross-react with the cellular 58 K protein. Furthermore, muscle did not exhibit any immunologically recognizable 58K protein. The antibody raised against mouse liver 58K recognized a similar protein in rat liver, kidney and testis. The 58K selenium-binding protein appeared to be concentrated in tissues enriched in G1 and the effect of hydroxyurea on selenium-binding protein levels suggested the 58K selenium-binding protein appeared to be made predominantly during the G1 phase of the cell cycle. Evidence is also presented that whole blood contains selenium bound to hemoglobin which supports prior evidence by other investigators. These results further support the evidence for tissue selenium-binding proteins other than glutathione peroxidase and document the immunological specificity and reactivity of a new antibody against a selenium-binding protein.