The equilibrium binding mechanism and kinetics of binding of diS-C3-(5) (3,3'-dipropylthiodicarbocyanine iodide) to rabbit renal brush-border membrane vesicles (BBMV) were examined using steady-state and time-resolved fluorescence, and fluorescence stopped-flow methods. In aqueous solution, diS-C3-(5) exists as a monomer at concentrations less than 5 microM with fluorescence emission peak at 670 nm (excitation 622 nm), anisotropy r = 0.102, and lifetime tau = 1.2 nsec (23 degrees C). Upon addition of increasing BBMV (voltage clamped to 0 mV using K+/valinomycin), the 670 nm emission peak decreases, corresponding to formation of a nonfluorescent membrane dimer, and subsequently a new emission peak at 695 nm increases, corresponding to membrane monomer. Dynamic depolarization studies show that aqueous diS-C3-(5) rotation is unhindered with a rotational rate R = 0.57 nsec-1 while membrane monomer is hindered with steady-state anisotropy r = 0.190, lifetime tau = 2.1 nsec, R = 0.58 nsec-1 and limiting anisotropy r infinity = 0.11. Based on equilibrium fluorescence titrations, the membrane monomer-dimer (M-D) dissociation constant, Kd = [M]2/[D][BBMV], is 0.0013, where BBMV is expressed as membrane phospholipid concentration. Three distinct kinetic processes are identified by stopped-flow experiments in which BBMV are mixed with diS-C3-(5). There is rapid binding of diS-C3-(5) to the membrane to form bound monomer with a 6-msec exponential time constant. The membrane monomer at the membrane outer surface then aggregates to form bound dimer at the outer surface with a concentration independent time constant of 30 msec. The overall dimerization reaction probably consists of a rate-limiting reorientation process (30 msec) followed by a rapid dimerization which occurs on a nanosecond time scale. Finally, there is a 0.8 to 1 sec translocation of membrane dimer between symmetric sites at the inner and outer membrane surfaces. The translocation reaction is the step which is probably sensitive to changes in transmembrane electrical potential.