The hydrolysis of cellooligosaccharides with a degree of polymerization (DP) up to 8 by cellobiohydrolases (CBH) I and II from Trichoderma reesei MCG 77 was investigated. Both enzymes degraded the oligomeric substrates according to a "multiple attack" mechanism without release of significant amounts of intermediate hydrolysis products with a DP higher than 3. CBH I was determined not to be a real 1,4-beta-D-glucan-cellobiohydrolase because glucose was produced from all oligomeric substrates. The maximum initial velocity of substrate degradation by CBH I was highest for DP 6. K-m values decreased from DP 4 to 6 and remained almost constant for higher DPs, indicating that the active site of CBH I spans at least six glucosyl-units. Based on the known three-dimensional structure of the core protein of CBH II, the product ratio of cellobiose to cellotriose released by the action of CBH II on different cellooligosaccharides could be explained and theoretically predicted. A beta-glycosidic bond at the nonreducing end of the substrate was found to be necessary for recognition by CBH I as well as CBH II because alpha-D-glucosyl-(1,4)-cellopentaose was not degraded by both enzymes. A mathematical model based on a reaction-rate-dependent, reversible loss of active enzyme, interpreted in terms of nonproductive substrate binding, was derived. This non-steady-state model was valid to predict concentration-time course data for the hydrolysis of all oligomeric substrates by CBH I and CBH II very well. The time-dependent substrate degradation and product formation clearly deviated from integrated Michaelis-Menten kinetics.