Heme has been receiving considerable interest as a prosthetic group of ribozymes and deoxyribozymes, because heme-bound nucleic acids exhibit peroxidase-like catalytic activities (Travascio, P., Li, Y., and Sen, D. (1998) Chem. Biol, 5, 505-517). The interaction of heme with dimeric G-quadruplexes formed from d(TAGGGTTAGGGT) and d(TAGGGTTAGGGA) has been characterized to gain a deeper understanding of the molecular recognition of G-quadruplex DNAs by heme. We found that heme binds selectively to the 3'-terminal G-quartet of a dimeric parallel G-quadruplex of d(TAGGGTTAGGGT), whereas binding of heme to a dimeric antiparallel G-quadruplex of d(TAGGGTTAGGGA) does not occur, suggesting that an orderly arrangement of the constituent guanine deoxyribose rings, with respect to the G-quartet plane, is crucial for the binding of heme to the DNA. The preferential binding of heme to the 3'-terminal G-quartet of parallel G-quadruplex DNAs allowed a systematic modification of the heme environment in the complex through the DNA sequence. The activity of the complexes was found to increase with increasing number of adenine bases adjacent to the heme in the complexes, possibly due to improvement of the accessibility of aromatic substrate, i.e., 10-acetyl-3,7-dihydroxyphenoxazine, to the heme, and an increase in the frequency of appearance of a specific orientation of the adenine bases, with respect to the heme, optimized for its activity as an acid-base catalyst to enhance the peroxidase activity of the complex.