In the present study, steady-state, picosecond time-resolved fluorescence and polarization gated anisotropy have been used to establish simultaneous binding of an intercalator (ethidium bromide, EtBr) and a minor groove binder (Hoeschst 33258, H258) to a dodecamer DNA of specific sequence. The Forster resonance energy transfer (FRET) studies between the dyes H258 (donor) and EtBr (acceptor) in the dodecamer, where the ligands have a particular relative orientation of the transition dipoles, in contrast to the cases in sodium dodecyl sulfate (SDS) micelles and larger genomic DNA, where the orientations are random, reveal the effect of the binding geometry of the ligands in the constrained environment. Our study establishes that reconsideration of the value of the orientation factor (kappa(2)) is crucial for correct estimation of the donor-acceptor distance when the ligands are simultaneously bound to a specific region of biological macromolecules.