Self-assembled monolayers (SAMs) of carboxylate- and amine-terminated alkanethiols were formed on gold-nanoparticle-coated microspheres. The stability and place-exchange reactions of fluorescently labeled derivatives of these monolayers were studied as a function of time and storage conditions. Changes in the fluorescence intensity of the derivatized SAMs were monitored using fluorescence microscopy. Gold-nanoparticle-coated microspheres were prepared by first derivatizing silica microspheres with a thiol-containing silane followed by self-assembly of the gold nanoparticles onto the microsphere surfaces. Nanoparticle assembly was performed by mixing thiol-activated silica with a citrate-stabilized solution of gold nanoparticles in ultrapure water. The mean diameter of the gold particles was 14.5 +/- 0.9 nm as determined by transmission electron microscopy (TEM). The mean diameter of the gold nanoparticles, after assembly onto the microsphere surfaces, was essentially unchanged (14.0 +/- 2.8 nm). The relative surface coverage of the silica microspheres with gold was found to be dependent on the concentration of gold nanoparticles in solution and on the incubation time. Field-emission scanning electron microscopy (FE-SEM) was used to obtain high-resolution images of the microspheres before and after the gold coating procedure. As the surface coverage increased, the measured surface roughness of the silica microspheres also increased. Tapping mode atomic force microscopy (AFM) was used to measure the surface roughness of individual microspheres. The surface roughness of the microspheres and the chemical composition of the SAM were found to correlate with differences in SAM stability. SAMs on microspheres with relatively higher surface roughness were less stable and were more susceptible to place-exchange reactions. Carboxylate-terminated monolayers were found to be more stable than amine-terminated SAMs. A novel method for determining the relative amounts of exchange of fluorescently labeled alkanethiols between different monolayer-coated microspheres is reported. In this method, the transfer of fluorescently labeled alkanethiols composing SAMs on one set of gold-coated microspheres to nonlabeled SAMs on another set of gold-coated microspheres is monitored as a function of time using fluorescence microscopy. A collision-dependent mechanism was found to influence the rate and amount of exchange of alkanethiol between microspheres. The stability and exchange properties of SAMs on gold-nanoparticle-coated microspheres are presented in this paper.