We describe physicochemical properties in DNA of altered-size nucleobases that retain Watson−Crick analogous hydrogen-bonding ability. Size-expanded analogues of adenine and thymine (xA and xT, respectively, which are expanded by benzo-fusion) were incorporated into natural DNA oligonucleotides, and their effects on helix stability were measured. Base stacking studies revealed that the two stretched analogues stack much more strongly than do their naturally sized counterparts. In contrast to this, pairing studies showed that single substitutions of the new bases are destabilizing to the natural helix as compared to A or T in standard A−T pairs in the same context, unless multiple adjacent substitutions are used. Interestingly, the size-expanded bases displayed selective recognition of the hydrogen-bonding complementary partners, suggesting that Watson−Crick analogous pairs were still formed despite local backbone strain. In an attempt to compensate for the added size of the expanded adenine, we tested a formamide deoxynucleoside, which Leonard proposed as a shortened thymine analogue (F_o). Data showed, however, that this compound adopts a conformation unfavorable for pairing. On the basis of the combined thermodynamic data, we estimate the energetic cost of the 2.4 Å stretching of an isolated base pair in DNA at ca. +1 to 2 kcal/mol. Notably, during the pairing studies, the two size-expanded nucleobases were found to display significant changes in fluorescence on formation of stacked versus unstacked structures, suggesting possible applications in probing nucleic acid structures and biochemical mechanisms.