Pseudoisocytidine, a C-nucleoside analogue of cytosine, has two possible isomers of the H1- and H3-forms. Enzymatic incorporation experiments confirmed the existence of the two isomers in solution, and the 2′-deoxyribonucleoside triphosphate of pseudoisocytosine (PIC) was incorporated into DNA opposite both guanine and 6-methoxypurine (M) by the Klenow fragment of Escherichia coli DNA polymerase I. In addition to the PIC·M pairing in replication, M also functioned as an A analogue and T was efficiently incorporated opposite M. Thus, the PIC·M pair is regarded as a base pair between a C analogue and an A analogue, and can mediate the interconversion between the G·C and A·T base pairs. The combination of PIC and M could be used as a G·C↔A·T transition mutagen. © 2002 Elsevier Science Ltd. All rights reserved.