Ultraviolet hyperchromicity experiments indicate that in DNA duplex formation, a C–T mismatch is destabilizing in the center of a duplex, but behaves as a stable base pair at the terminus of a duplex. The C–T base pair is thought to contain two hydrogen bonds, but has thermodynamic parameters (ΔH° and ΔG° of dissociation) that are similar to a G‐C base pair. AMBER molecular mechanics calculations were performed to study the possible structural properties of DNA duplexes with central and terminal C–T combinations. These calculations also indicate that a central C–T pair destabilizes a duplex, while terminal C–T forms a stable base pair. Hydrogen bonding between cytosine and thymine occurs only in the energy‐minimized structures when the helix diameter decreases and the propeller twist angle between the bases increases. These changes are found to occur only at the end of a duplex in the calculations, which may explain the experimental results.