The unsymmetrical cyanine dyes BOXTO-PRO and BOXTO-MEE were derived from the DNA groove binder BOXTO, by adding a positively charged or a non-ionic hydrophilic tail to BOXTO, respectively. The main objective was to obtain more efficient DNA probes, for instance in electrophoresis and microscopy, by slowing down the dissociation of BOXTO from DNA. The interactions with mixed sequence DNA was studied with fluorescence and absorbance spectroscopy, stopped-flow dissociation and gel electrophoresis. Both the derivatives are groove bound as BOXTO, and have similar fluorescence properties when bound to mixed sequence DNA in free solution. BOXTO-PRO exhibits a slower dissociation than BOXTO from DNA, whereas the dissociation rate for BOXTO-MEE is faster and, unexpectedly independent of the ionic strength. During gel electrophoresis both BOXTO-PRO and BOXTO-MEE exhibit a faster dissociation rate than BOXTO. Still, BOXTO-PRO seems to be a good alternative as DNA probe, especially for applications in free solution where the dissociation is slower than for the corresponding intercalator TOPRO-1.