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Invariant chain (li) is a nonpolymorphic glycoprotein that associates with major histocompatibility complex class II molecules and has been shown to mediate several functions in class II-restricted antigen presentation. A small proportion of li is modified by the addition of chondroitin sulfate (li-CS), and this form of li is associated with class II on the surface of antigen-presenting cells. In this report we show that expression of li-CS dramatically enhanced the ability of class II-positive EL4 transfectants to stimulate class II-dependent allogeneic and mitogenic T cell responses. Antibody blocking studies and the ability of CD44 to bind directly to li-CS suggest that li-CS can function as an accessory molecule during T cell responses through interactions with CD44.