Chondroitin 6-sulfotransferase (C6ST) catalyzes the transfer of sulfate to position 6 of the N-acetylgalactosamine residue of chondroitin. To obtain direct evidence regarding the function of C6ST and its product, chondroitin 6-sulfate, in vivo, we isolated the mouse C6ST gene (C6st) and generated mice deficient in this gene (C6st-/-) by embryonic stem cell technology. C6st-/- mice were born at approximately the expected frequency and were viable through adulthood. In the spleen of C6st-/- mice, the level of chondroitin 6-sulfate became almost undetectable. Analyses of these knockout mice provided insights into the biosynthesis of oversulfated chondroitin sulfates in mice; chondroitin sulfate D in the brain of null mice and the cartilage and telencephalon of null embryos disappeared, while the chondroitin sulfate E level in the spleen and brain of the null mice was unchanged. In spite of the disappearance of chondroitin sulfate D structure, brain development was normal in the C6st-/- mice. Further analysis revealed that the number of CD62L+CD44low T lymphocytes corresponding to naive T lymphocytes in the spleen of 5-6-week-old C6st-/- mice was significantly decreased, while those in other secondary lymphoid organs were unchanged. This finding suggested that chondroitin 6-sulfate plays a role in the maintenance of naive T lymphocytes in the spleen of young mice.