We have the studied the binding of 5-((N-(5-(N-(6-(biotinoyl)amino)hexanoyl fluorescein (fluorescein biotin) to 6.2 mu m diameter, streptavidin-coated polystyrene beads using a combination of fluorimetric and flow cytometric methods. We have determined the average number of binding sites per bead, the extent of fluorescein quenching upon binding to the bead, and the association and dissociation kinetics. We estimate the site number to be approximate to 1 million per bead. The binding of the fluorescein biotin ligand occurs in steps where the insertion of the biotin moiety into one receptor pocket is followed immediately by the capture of the fluorescein moiety by a neighboring binding pocket; fluorescence quenching is a consequence of this secondary binding. At high surface coverage, the dominant mechanism of quenching appears to be via the formation of nonfluorescent nearest-neighbor aggregates. At early times, the binding process is characterized by biphasic association and dissociation kinetics which are remarkably dependent on the initial concentration of the ligand. The rate constant for binding to the first receptor pocket of a streptavidin molecule is approximate to(1.3 +/- 0.3) x 10(7) M-1 s(-1). The rate of binding of a second biotin may be reduced due to steric interference. The early time dissociative behavior is in sharp contrast to the typical stability associated with this system. The dissociation rate constant is as high as 0.05 s(-1) shortly after binding, but decreases by 3 orders of magnitude after 3 h of binding. Potential sources for the time dependence of the dissociation rate constant are discussed.