We explore the relationship between the electronic-nuclear level structure, the electronic couplings, and the dynamics of hole hopping transport in DNA. We utilized the electronic coupling matrix elements for hole transfer between nearest-neighbor nucleobases in DNA [Voityuk, A. A.; Jortner, J.; Bixon, M.; Rösch, N. J. Chem. Phys. 2001, 114, 5614] to evaluate intrastrand and interstrand superexchange electronic couplings, which determine hole hopping rates within the framework of a semiempirical quantum mechanical-kinetic model. Calculations of the exponential distance (R) dependence of the superexchange mediated intrastrand electronic couplings |Vsuper|2 exp(−βR) between guanines (G) in “short” G+(T−A)nG (n 3) duplexes result in β = 0.8−0.9 Å-1. We interpret the experimental data on time-resolved hole transport in the presence of a site-specifically bound methyl transferase mutant in DNA [Wagenknecht, H.-A.; Rajski, S. R.; Pascally, M.; Stemp, E. D. A.; Barton, J. K. J. Am. Chem. Soc. 2001, 123, 4400] in terms of composite sequential, interstrand and intrastrand superexchange mediated, and direct interstrand hole hopping. This mechanism accounts for the rate determining step, for the weak duplex size dependence of the rate, and for the long-range charge transport induced by interstrand superexchange via short (T−A) bridges, containing a single mediating nucleobase. For hole transfer via longer (T−A)n (n 3) bridges, the superexchange mechanism is replaced by the parallel mechanism of thermally induced hole hopping (TIH) via long (A)n chains. A kinetic analysis of the experimental data for hole transport through seven GG pairs separated by (T−A)n (n = 2−5) bridges across the 3‘−5‘ strand of the DNA duplex [Sartor, V.; Boone, E.; Schuster, G. B. J. Phys. Chem. B, 2001, 105, 11057] reveals that the superexchange−TIH crossover occurs at n = nx = 3. The explorations of the range of applicability and the breakdown of the superexchange mechanism in DNA lay the foundations for the scrutiny of the universality and system specificity of this mechanism in large-scale chemical and biophysical systems.