Lanatoside C, a digitalis glycoside, has previously been shown to be a potent polyclonal B cell activator for mouse lymphocytes. Since the chemical structure of lanatoside C is well defined we investigated the effect of different chemical modifications on the mitogenic properties of the glycoside in order to determine the molecular basis of B cell activation. The presence of an OH- group at C12 in the steroid nucleus proved to be essential for activation to occur in serum-free medium. Likewise, an intact carbohydrate side chain at C3 appeared to be necessary for retaining mitogenic properties. In serum-supplemented cultures, however, a much more complex response pattern was observed where the prerequisites for activation were not as distinct as in serum-free medium.