The conformational properties of Rhodobacter capsulatus cytochrome c2 wild-type and two site-directed mutants (glycine 34 replaced by serine and proline 35 replaced by alanine) were characterized by their charge state distributions and hydrogen/deuterium (H/D) exchange properties monitored by electrospray ionization mass spectrometry. The results suggest the presence of structural perturbations in the mutated cytochromes, an observation that is in agreement with their decreased conformational stabilities. In addition, a fast enzymatic procedure was developed to identify regions for which the H-bonding or solvent accessibility properties were perturbed by the mutations. In this procedure, deuterated peptides were separated and analysed by using liquid chromatography directly coupled to the electrospray ionization source in order to minimize the occurrence of back-exchange during analysis. In the case of G34S, mutational effects were found for peptides 1–26, 38–51, 52–59 and 109–116, which in the Rb. capsulatus cytochrome c2 structure correspond to extensive regions on the same side of the molecule as the proximal histidine, as well as part of the C-terminal helix. In the case of P35A, mutational effects were found for peptides 1–26, 27–37, 38–51 and 52–59, which in the Rb. capsulatus cytochrome c2 stucture correspond to extensive regions on the same side of the molecule as the proximal histidine. We show that the present set of mass spectrometric experiments is useful as an initial characterization of mutant conformational properties because the analyses require only nanomole quantities of protein and can be performed rapidly.