CD223 (LAG-3) is an activation-induced cell surface molecule, structurally similar to the T cell coreceptor CD4, that binds MHC class II molecules with high affinity. Little is known about the expression and function of murine CD223. Here, we show that mRNA expression is restricted to the thymic medulla, splenic red pulp and sparse cells in the adult brain cortex. In contrast, surprisingly high expression was seen in defined tracts at the base of the cerebellum and in the choroid plexus of day 7 postnatal brain. mCD223:Ig, but not CD4:Ig, fusion proteins stained cells expressing MHC class II molecules. Analysis of mCD223 cell surface expression was performed with a new monoclonal antibody (mAb) that recognizes an epitope in the D2 domain. Although it blocked mCD223 function in vitro, it did not block binding of mCD223 to MHC class II molecules. While very few TCRαβ T cells in the spleen and thymus of naive mice express surface mCD223 (<3%),∼ 18% TCRγδ T cells and ∼ 10% NK cells are positive. This small population of TCRαβ T cells are cycling memory T cells (BrdU+, CD44hi, CD62Llo). In contrast, all T cells express mCD223 2-3 days post activation. This study and the anti-CD223 mAb should greatly assist in the elucidation of CD223 function.