MHC class II molecules, composed of alpha- and beta-chain heterodimers, are required for Ag presentation. The carboxyl-terminal domains of class II molecules are believed to mediate the location of class II in the plasma membrane and are important for signal transduction and Ag presentation. These domains contain typical transmembrane sequences, and cytoplasmic sequences of 12 or 18 amino acids for the alpha- and beta-chains, respectively. We examined these domains to determine whether they linked class II molecules to the actin-based cytoskeleton. Our analyses of class II-cytoskeleton interactions, such as a colocalization with actin filaments during capping, association with the detergent-insoluble cytoskeleton, and direct binding of filamentous actin, revealed that both the cytoplasmic and transmembrane domains contributed to class II interactions with the cytoskeleton. Detergent-extracted and immunoprecipitated full-length class II molecules had quantitatively stronger interactions with the cytoskeleton than did molecules with deleted cytoplasmic domains. A secondary Ab, which was used to cross-link primary Ab bound to class II, up-regulated the class II-cytoskeletal associations. This association was efficiently inhibited by dihydrocytochalasin B, but only partially disrupted by chlorpromazine. The mechanism of interaction with actin filaments after ligation of class II occurred without a measurable increase in filamentous actin levels. This suggested that enhanced class II-cytoskeleton associations involved a rearrangement of existing actin filaments, possibly through the multiple kinases that are activated after class II transmembrane signaling.