The common nitrogen mustard, mechlorethamine, can form a covalent cross-link between the two bases of a cytosinecytosine mismatch pair within a DNA duplex. The cross-linked species can be readily separated from DNA monoadducts and unreacted strands using denaturing polyacrylamide gel electrophoresis. Here, using DNA 19 mer duplexes that are mechlorethamine cross-linked at a C4C35, C7C32, C10C29, or C13C26 mismatch pair, we show that the denaturing polyacrylamide gel electrophoresis mobility of the cross-linked species is particularly sensitive to the proximity of the CC cross-link to the duplex end. Species that are cross-linked at a C4C35 mismatch have greater mobilities than those cross-linked at C7C32 or C13C26, and the species with a central C10C29 cross-link have the lowest mobility. The mobility is also dependent on the proximity of the cross-link to a 5-32P-phosphate or a 5-fluorescein label. We interpret these results in terms of the conformational properties of the cross-linked species in the denaturing gel. The results are consistent with the retention of partial duplex character at the end proximal to the cross-link, with an influence on the mobility of the GC/AT ratio proximal to the cross-link and at the duplex end, and a small but discernible effect of the label.