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 40 mM NaHCO3 (pH 8.0) buffer depend upon the free dye concentration with the reaction taking several minutes to reach completion even in relatively high, 40 nM, 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 x 10(5) S-1 M-1, the dissociation constant to be 12.1 +/- 3.4 nM and the maximum DNA molecule extension to be 36 +/- 4%. The intercalation time constant (inverse of the pseudo-first order rate constant), tau, decreased from 309 to 62 s as YOYO-1 levels increased from 10 to 40 nM. 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. (C) 2010 Elsevier Inc. All rights reserved.