We have investigated energy transfer in a novel self-assembled DNA hybrid structure composed of diaminopurine-equipped naphthalene derivatives that are hydrogen-bonded along a single-stranded oligothymine template. By performing time-resolved measurements of the naphthalene donor luminescence decay in the absence and presence of a cyanine Cy3.5 acceptor bonded covalently to the 5' end of the oligothymine, we have examined the role of temperature and DNA template length on energy transfer from donors to the acceptor. We find that energy transfer rates decline with increasing temperature over a fairly narrow (±5 °C) range over which changes in circular dichroism and donor luminescence lifetime indicate that the chiral assemblies are dissociating. In addition, the transfer rates exhibit a complex dependence on template length, increasing from initially low values for 10 bases toward an optimum for 30 bases and then declining again toward 60 bases. We find that for short (10 bases) templates, incomplete filling and disorder reduces the overall transfer efficiency, while longer assemblies are more ordered but suffer from larger donor–acceptor separations, resulting in the observed peak at intermediate template length. In order to replicate the observed transfer dynamics, we have constructed a model assuming Fo¨rster energy transfer occurs between donors and acceptors whose geometric arrangement had been determined through molecular dynamics simulations of the whole assembly structure. For short DNA templates, the model is found to overestimate the transfer rates because it does not include effects of incomplete complex assembly and stacking faults. In contrast, the model underestimates the transfer rates for long, ordered assemblies indicating that additional mechanisms, such as diffusion of excitations along the donor stacks, need to be included. These results suggest that efficient energy transfer, in excess of that expected from simple Fo¨rster calculations, is feasible even for long DNA-templated assemblies of p-stacked conjugated chromophores. Such structures may therefore act as molecular wires transporting energy from one end to another.