We here exploit the recently reported thermodynamic preference for poly(dAdT)(2) over mixed-sequence calf thymus (ct) DNA of two binuclear ruthenium complexes, Delta Delta-[mu-bidppz(bipy)(4)Ru-2](4+) (B) and Delta Delta-[mu-bidppz(phen)(4)Ru-2](4+) (P), that bind to DNA by threading intercalation, to determine their intrinsic dissociation rates. After adding poly(dAdT)(2) as a sequestering agent to B or P bound to ct-DNA, the observed rate of change in luminescence upon binding to the polynucleotide reflects the rate of dissociation from the mixed sequence. The activation parameters for the threading and dissociation rate constants allow us for the first time to characterize the thermodynamics of the exceedingly slow threading intercalation equilibrium of B and P with ct-DNA. The equilibrium is found to be endothermic by 33 and 76 kJ/mol, respectively, and the largest part of the enthalpy difference between the complexes originates from the forward threading step. At physiological temperature (37 degrees C) B and P have dissociation half-lives of 18 and 38 h, respectively. This is to our knowledge the slowest dissociating noncovalently bound DNA-drug reported. SDS sequestration is the traditional method for determination of rate constants for cationic drugs dissociating from DNA. However, the rates may be severely overestimated for slowly dissociating molecules due to unwanted catalysis by the SDS monomers and micelles. Having determined the intrinsic dissociation rates with poly(dAdT)(2) as sequestering agent, we find that the catalytic effect of SDS on the dissociation rate may be up to a factor of 60, and that the catalysis is entropy driven. A simple kinetic model for the SDS concentration dependence of the apparent dissociation rate suggests an intermediate that involves both micelles and DNA-threaded complex.