The thermodynamics and folding kinetics of mutants at the helix N-terminus and hydrophobic core of Chymotrypsin Inhibitor 2 (CI2) have been studied. All mutants adhere to a two-state model for protein folding , and are destabilised relative to wild-type. Mutation of N-cap residue S31 to Ala or Gly destabilises CI2 by nearly 1 kcal mol-1, with respect to both wild-type and the double mutant EA33EA34. Mutation of E33 or E34 to Gin, Asp and Asn progressively destabilises the protein from 0.3 -

1. I kcal mol- 1. Deletion of one methyl(ene) group from the hydrophobic core of CI2 destabilises the protein on average by 1.3 kcal mol-1, with a strong correlation between the environment of the mutation and its effect on stability. Finally, the helix N-terminus and hydrophobic core are partially formed in the transition state of CI2, with increased exposure to solvent compared to the native state.