This paper describes attempts to increase the kinetic stability of chitinase B from Serratia marcescens (ChiB) by the introduction of semi-automatically designed rigidifying mutations of the Gly-->Ala and Xxx-->Pro type. Of 15 single mutants, several displayed significant increases in thermal stability, whereas most mutants showed minor effects. All mutations with non-marginal effects on stability clustered in a limited, surface-exposed region of the enzyme, indicating that this region is involved in a partial unfolding process that triggers irreversible thermal inactivation (aggregation). A double mutant containing two stabilizing mutations in this region (G188A, A234P) displayed a 10-fold increase in half-life at 57 degrees C and a 4.2 degrees C increase in apparent T(m). These results show that entropic stabilization works well for ChiB and they pinpoint a region whose unfolding may be crucial for the kinetic stability of this enzyme.