Beside the known maximum of electron capture dissociation (ECD) of gas-phase polypeptide polycations at zero electron energy, a broad local maximum is found around 10 eV. This maximum is due to electronic excitation prior to electron capture, as in dissociative recombination of small cations. In the novel hot electron capture dissociation (HECD) regime, not only N-C-alpha bonds are cleaved as in ECD, but secondary fragmentation is also induced due to the excess energy. Beneficially, this fragmentation includes abundant losses of .CH(CH3)(2) from leucine and .CH2CH3 from isoleucine residues terminal to the cleavage site, which allows for distinguishing between these two isomeric residues. (C) 2002 Elsevier Science B.V. All rights reserved.