Activation of the enediyne neocarzinostatin chromophore (NCS-Chrom) by thiol addition at C-12 generates a diradical species with radical centers at C-2 and C-6, which abstract hydrogens from deoxyribose in the minor groove of DNA. Since hydrogen abstraction from DNA accounts for only part of the hydrogen incorporated at these sites, it is important to determine the other possible sources. At low concentration of thiol, a condition resembling that during NCS-Chrom-induced DNA damage, the major non-DNA hydrogen donation source was found to be the carbon-bound hydrogen of the aqueous methanol solvent, rather than the expected sulfur-bound hydrogen of the thiol. Further, experiments with the gamma-L-glutamyl-DL-cysteinylglycine labeled with deuterium on the alpha- or beta-carbons to the sulfur showed small amounts of internal transfer of hydrogen into C-2 of the drug from the naturally occurring L,L diastereomer only. Quantitation of the hydrogen transfer was accomplished by separation of the L,DL diastereomeric mixtures of the thiol-NCS-Chrom adducts. In all, these various hydrogen donation sources can account for at least 70-80% of the hydrogen incorporated at C-2 of the drug under DNA damage conditions. Selective quenching of the radical at C-2 could account for the predominance of single-stranded over double-stranded DNA lesions.