Clostridium thermocellum cellulosomes isolated by cellulose affinity chromatography were fractionated by anion exchange chromatography into apparently homogeneous subpopulations that differed with respect to enzyme activity and subunit composition. One such subpopulation contained predominantly six subunits and was closely similar to the "subcellulosome" described by Kobayashi et al. (Kobayashi, T., Romaniec, M. P. M., Fauth, U., and Demain, A. L., Appl. Environ. Microbiol., 1990, 56, 3040-3046). Avicelase specific activity of this homogeneous subpopulation was slightly higher than that of unfractionated cellulosomes, but the two preparations were similarly affected by Ca2+, dithiothreitol, and cellobiose. Determination of their N-terminal sequences and enzyme activities has enabled three of the six major subunits of the subpopulation of cellulosomes to be positively identified as known components of the C. thermocellum cellulase complex; the other three subunits did nor match up with previously characterized cellulosomal proteins.