The synthesis of two novel PNA nucleobases derived from 1,8-naphthyridin-2(1H)-one (bT) and benzo[b]-1,8-naphthyridin-2(1H)-one (tT) are reported, together with their incorporation into oligomers of PNA and evaluation as substitutes for thymine. Compound bT is shown to be an effective mimic of the natural thymine nucleobase in PNA-DNA, PNA-RNA, and PNA-PNA duplex structures. A study using singly mismatched target sequences showed bT to be selective for the recognition of adenine. The X-ray structure of a PNA hexamer containing a single bT base (H-GbTATAC-L-lys-NH2) was determined to 1.8 A resolution and confirmed the base-pairing capability with adenine. The introduction of a bT base does not alter the P-form double helix structure of PNA, as compared to other PNA structures containing natural nucleobases. With the tricyclic derivative, incorporation of several units of tT resulted in decreased specificity in some systems, while maintaining specificity in others. In PNA·DNA-PNA triplex structures, incorporation of bT into the Hoogsteen strand resulted in enhanced stability relative to control triplexes containing only T·A-T and pseudoisocytosine (J)·G-C triplets (ΔTm = +1.5 °C/modification). The evaluation of another nucleobase, 3,5-diaza-4-oxophenothiazine (tC), expected to mimic the function of cytosine is similarly reported. Incorporation of tC in place of cytosine in PNA oligomers increased the thermal stability of the corresponding PNA-DNA, PNA-RNA, and PNA-PNA duplexes. However, the sequence specificity was diminished in some PNA-DNA duplex systems containing several tC units. The thermal stability of triplex structures containing tC in the Hoogsteen strand was reduced relative to the cytosine-containing control.