We present results of a two-photon fluorescence-correlation study carried out with glycosylated and untreated 20 nm fluorescing spheres that interacted with the carbohydrate-binding proteins soybean agglutinin (SBA) and concanavalin A (Con A). The assay principle allows protein-carbohydrate binding interactions to be determined without protein labeling. This assay might serve as a simple model system for studying physical and chemical interactions between proteins and carbohydrates, for example, at cell or virus surfaces. In experiments with galactosylated 20 nm beads and SBA, several stages of protein-carbohydrate interactions could be clearly distinguished. Initially, only a few lectins bound to the nanospheres. At higher lectin concentrations polymerization occurred, and aggregates consisting of about 2.6 x 10(5) glycosylated nanospheres were formed. At very high lectin concentrations, the degree of polymerization dropped, and the size of single SBA-covered nanospheres increased to approximately 40 nm. When Con A was used instead of SBA, a significantly smaller degree of aggregation (4 x 10(4) spheres) was obtained. Treatment of unglycosylated 20 nm beads with SBA as a negative control sample resulted in a much lower unspecific aggregation (5 x 10(3) spheres). The assay principle can thus help to elucidate relative binding affinities.