The cyanine dye, YOYO-1, has frequently been used in single DNA molecule imaging work to stain double-stranded DNA as it fluoresces strongly when bound. The binding of YOYO-1 lengthens the DNA due to bis-intercalation. We have investigated the kinetics of binding, via this increase in DNA length, for single, hydrodynamically-stretched molecules of lambda DNA observed via Total Internal Reflection Fluorescence (TIRF) microscopy. The rate and degree of lengthening in 40mM NaHCO 3 (pH 8.0) buffer depend upon the free dye concentration with the reaction taking several minutes to reach completion even in relatively high, 40nM, concentrations of YOYO-1. In the absence of overstretching of the DNA molecule, we determine the second order rate constant to be 3.8±0.7×10 5 s −1 M −1 , the dissociation constant to be 12.1±3.4nM and the maximum DNA molecule extension to be 36±4%. The intercalation time constant (inverse of the pseudo-first order rate constant), τ, decreased from 309 to 62s as YOYO-1 levels increased from 10 to 40nM. The kinetics of binding help with interpretation of the behavior of DNA–YOYO-1 complexes when overstretched and establish defined conditions for the preparation of DNA–YOYO-1 complexes.