Ab initio quantum mechanical calculations were employed to examine the binding energies and geometries of several nonnatural base analogues of nucleic acid bases bound to the major groove of the G.C base-pair in an attempt to explore promising candidate bases for triplex-helix-forming oligonucleotides. Seven neutral base analogues together with guanine and protonated forms of cytosine and adenine are considered. The full geometry optimizations were carried out at the HF/6-31G(d) level, and single-point energies were obtained at the MP2/6-31 G(d,p)//HF/6-31G(d) level. Our analysis reveals that protonated cytosine (C+) has the highest binding energy over the other bases. C-5-methylation of protonated cytosine does not improve the base-pairing efficiency over protonated cytosine. Among the neutral bases, the binding efficiency of 6-thioguanine is the closest to that of guanine in the Py*Pu.Py type motif (1.4 kcal/mol less than guanine at MP2/6-31G(d,p)//HF/6-31G(d)), and 8-oxoadenine ranked the least. Semiempirical AM1 and PM3 methods compare qualitatively well with the energetic trends of that predicted by the HF/6-31G(d) method. At the MP2/6-31G(d,p)/HF/6-31G(d) (BSSE and ZPE corrected) level, the following order of major-groove binding affinity is established. C+*G.C > C-5M(+)*G.C > A(+)*G.C > G*G.C > CX1*G.C > (6-thio)G*G.C > (8-thio)A*G.C > C*6-oxo*G.C > C-5M,C-6-oxo*G.C > (8-oxo)A*G.C. Close consideration of the structural isomorphism of these triplexes with native C+*G.C and T*A.T and the major-groove binding efficiency of the bases suggests that the neutral form of 8-thioadenine ((8-thio)A) may be considered as an alternative to protonated cytosine for efficient triple-helix formation within the Py*Pu.Py type parallel motif.