The stability and replication of DNA containing self-pairs formed between unnatural nucleotides bearing benzofuran, benzothiophene, indole, and benzotriazole nucleobases are reported. These nucleobase analogues are based on a similar scaffold but have different hydrogen-bond donor/acceptor groups that are expected to be oriented in the duplex minor groove. The unnatural base pairs do not appear to induce major structural distortions and are accommodated within the constraints of a B-form duplex. The differences between these unnatural base pairs are manifest only in the polymerase-mediated extension step, not in base-pair stability or synthesis. The benzotriazole self-pair is extended with an efficiency that is only 200-fold less than a correct natural base pair. The data are discussed in terms of available polymerase crystal structures and imply that further modifications may result in unnatural base pairs that can be both efficiently synthesized and extended, resulting in an expanded genetic alphabet.