Cultured HeLa cells were incubated with pyrene-GM1/3H-radiolabeled GM1 ganglioside (1:4 M/M) mixtures for various times. The process of association of pyrene-GM1 with cells was qualitatively and quantitatively the same as that of 3H-GM1. The pyrene-GM1 and 3H-GM1 proportions in the various forms of association with cells were similar to that of the starting ganglioside mixture. After 2-h incubation, the association of ganglioside with cells was well established whereas almost no metabolic processing had occurred. During a 24-h incubation, pyrene- and 3H-GM1 underwent similar metabolic processing and gave rise to catabolic (GM2 and GM3) and anabolic (GD1a) derivatives. Fluorescence spectroscopy experiments carried out with the excimer formation technique on subcellular fractions containing plasma membranes showed that exogenous ganglioside was, in part, associated with the cells in a micellar form removable by trypsin treatment, and in part inserted in a seemingly molecular dispersion. Addition of Ca2+ salts caused aggregation of the ganglioside, as indicated by the increase of the excimer:monomer fluorescence ratio. The phenomenon was Ca2+ concentration dependent (maximum at 10 mM), and subsequent addition of EDTA had no effect. The saccharide portion of exogenously incorporated pyrene-GM1 was available to interact with external ligands, as shown by its ability to bind cholera toxin whose addition reduced the collision rate among the ganglioside lipid moieties.