Selenoprotein P (Sepp1) has two domains with respect to selenium content: the N-terminal, selenium-poor domain and the C-terminal, selenium-rich domain. To assess domain function, mice with deletion of the C-terminal domain have been produced and compared with Sepp1–/– and Sepp1+/+ mice. All mice studied were males fed a semipurified diet with defined selenium content. The Sepp1 protein in the plasma of mice with the C-terminal domain deleted was determined by mass spectrometry to terminate after serine 239 and thus was designated Sepp1Δ240–361. Plasma Sepp1 and selenium concentrations as well as glutathione peroxidase activity were determined in the three types of mice. Glutathione peroxidase and Sepp1Δ240–361 accounted for over 90% of the selenium in the plasma of Sepp1Δ240–361 mice. Calculations using results from Sepp1+/+ mice revealed that Sepp1, with a potential for containing 10 selenocysteine residues, contained an average of 5 selenium atoms per molecule, indicating that shortened and/or selenium-depleted forms of the protein were present in these wild-type mice. Sepp1Δ240–361 mice had low brain and testis selenium concentrations that were similar to those in Sepp1–/– mice but they better maintained their whole body selenium. Sepp1Δ240–361 mice had depressed fertility, even when they were fed a high selenium diet, and their spermatozoa were defective and morphologically indistinguishable from those of selenium-deficient mice. Neurological dysfunction and death occurred when Sepp1Δ240–361 mice were fed selenium-deficient diet. These phenotypes were similar to those of Sepp1–/– mice but had later onset or were less severe. The results of this study demonstrate that the C terminus of Sepp1 is critical for the maintenance of selenium in brain and testis but not for the maintenance of whole body selenium.