We have designed and synthesized DNA duplexes containing 5-dimethylaminocytosine (C-DMA) to investigate the effects of C(5)-substituted cytosine bases on the transfer and trapping of positive charge (holes) in DNA duplexes. Fluorescence quenching experiments revealed that a C-DMA base is more readily one-electron oxidized into a radical cation intermediate as compared with other natural nucleobases. Upon photoirradiation of the duplexes containing C-DMA, the photosensitizer-injected hole migrated through the DNA bases and was trapped efficiently at the C-DMA sites, where an enhanced oxidative strand cleavage occurred by hot piperidine treatment. The C-DMA radical cation formed by hole transfer may undergo specific hydration and subsequent addition of molecular oxygen, thereby leading to its decomposition followed by a predominant strand cleavage at the C-DMA site. This remarkable property suggests that the modified cytosine C-DMA can function as an efficient hole-trapping site in the positive-charge transfer in DNA duplexes.