Threading intercalation is an unusual DNA binding mode with significantly slower association and dissociation rates compared with classical intercalation. The latter has been shown to correlate well with cytotoxicity, and therefore, threading intercalating compounds are of great interest in the search for new DNA binding drugs. Thus, there is a need for better understanding of the mechanisms behind this type of binding. In this work, we have investigated the threading intercalation ability of the four stereoisomers of the AT-specific binuclear ruthenium complex [μ-dppzip(phen)(4)Ru(2)](4+) using different spectroscopic techniques. This complex contains an unsymmetrical bridging ligand consisting of a dipyridophenazine and an imidazophenanthroline ring system, in which the photophysical properties of the Ru-dipyridophenazine complex moiety make it possible to distinguish the intercalating part from the nonintercalating part. We have found that Δ geometry around the ruthenium on the intercalating dipyridophenazine moiety and Λ geometry on the nonintercalating imidazophenanthroline moiety is the optimal configuration for threading intercalation of this complex and that the chirality on the ruthenium of the nonintercalating half dominates the stereospecificity in the threaded state. This is the cause of the reversed enantioselectivity compared with the parent threading intercalating complex [μ-bidppz(phen)(4)Ru(2)](4+), in which the enantioselectivity is controlled by the chirality on the intercalating half. The differences in the interactions with DNA between the two complexes are most likely due to the fact that [μ-dppzip(phen)(4)Ru(2)](4+) has a slightly shorter bridging ligand than the parent complex.