Cotton fibers treated with liquid ammonia and sodium hydroxide were hydrolyzed with crude cellulase. The structural change in the fibers due to the cellulase treatment was examined in relation to the apparent affinity of Congo Red. The cellulose III crystalline structure collapsed and generated intermediate-molecular-ordered regions on cellulase treatment. Adsorption of Congo Red occurred on the crystallite surfaces of cellulose II and cellulose III that was transformed from cellulose II. The fiber, the dominant crystallite phase of which was cellulose III that was transformed from cellulose II, had a considerably increased apparent affinity after cellulase treatment. The intermediate structure on the crystallite surface was associated with the adsorption of Congo Red in the cases of cellulose II and cellulose III.