An analysis of the recently published genome sequence of Cytophaga hutchinsonii revealed an unusual collection of genes for an organism that can attack crystalline cellulose. Consequently, questions were being raised by cellulase scientists, as to what mechanism this organism uses to degrade its insoluble substrates. Cellulose, being a highly polymeric compound and insoluble in water, cannot enter the cell walls of microorganisms. Cellulose-degrading enzymes have therefore to be located on the surface of the cell wall or released extracellularly. The location of most cellulase enzymes has been studied. However, basic information on C. hutchinsonii cellulases is almost non-existent. In the present study, the location, formation and biosynthetic regulation of cellulases in C. hutchinsonii were demonstrated on different substrates. Various fractions isolated from C. hutchinsonii after cell rupture were assayed for carboxymethyl-cellulase activity (CMC). The cellulases were found to be predominantly cell-free during active growth on solka-flok, although 30% of activity was recorded on cell-bound enzymes. Relatively little CM-cellulase was formed when cells were grown on glucose and cellobiose. Apparently glucose or labile substrates such as cellobiose seem to repress the formation of CM-cellulase. These findings should provide some insight into possible hydrolysis mechanisms by C. hutchinsonii.