To clarify the hole-transfer mechanism in DNA and the factor controlling the hole-transfer rates, the kinetics of hole transfer in DNA was studied by monitoring the transient absorption of the pyrene (Py) radical cation (Py.+) during the pulse radiolysis of oligodeoxynucleotides (ODNs) conjugated with Py. By analyzing the transient absorption of Py.+ formed by the hole transfer from DNA to Py, the rate constants of the hole transfer in various sequences of DNA were determined. The rate constants of the hole transfer from the nearest guanine (G) to Py were weakly dependent upon the distance between Py and the nearest G, indicating the occurrence of the multistep hole transfer in DNA. In contrast, in the hole transfer where the rate-determining step was the single-step hole transfer between Gs, the rate was strongly dependent upon the distance between the Gs. Comparing the intervening nucleobases between Gs, the rate constants of the multistep hole transfer increased in the order G(.+)AG > G(.+)AC > G(.+)TG. These results showed that the hole transfer between Gs was effectively mediated when the bridge was A. The effect of multiple Gs (GGG) on multistep hole transfer was examined. The hole transfer from DNA to Py was slowed by the presence of the GGG site that was far away from Py, indicating that the multiple G worked as a hole trap site. Our results are discussed in the context of the previously reported theoretical and experimental results.