An accurate method for deriving molar absorptivity-temperature profiles applied to a set of single-stranded oligodeoxyribonucleotides shows that the undecamer CGAGTTTGACGp exists in a hairpin conformation involving Watson-Crick base pairing between the two terminal CG dinucleotides. The hairpin, which has a transition midpoint of 40 degrees C in 0.115 M Na+, is unusually stable in comparison with previously reported hairpins. A non-linear least squares analysis of the undecamer's profile in terms of a two-state equilibrium model indicates that the hairpin-to-coil transition occurs with an enthalpy change about twice that expected if only combinations of Watson-Crick base-paired stacking interactions are considered. The analogous hairpin structure (containing an identical CG/CG stem) assignable to the complementary strand CGTCAAACTCGp does not form above 0 degrees C. Measurements on the two undecamers indicate that variation in non Watson-Crick interactions within the loops of two similar hairpins can produce a difference in stability of at least 2.2 kcal/mol (25 degrees C, 0.115 M Na+), roughly equal to the amount contributed to a double helix by a 5'-CG-3'/5'-CG-3' base-paired stacking interaction.