By interacting with each others, the tetraspanins are thought to assemble a network of molecular interactions, the tetraspanin web. These tetraspanin/tetraspanin interactions involve in part the palmitoylation of the proteins. We show that tetraspanins interact with cholesterol as indicated by the precipitation of tetraspanin/tetraspanin complexes by digitonin, a cholesterol‐precipitating reagent, and the labeling of the tetraspanins CD9, CD81 and CD82 with a photoactivatable cholesterol in vivo. Cholesterol may participate to the interaction of tetraspanins with each other since digitonin‐precipitation of tetraspanins was correlated with their mutual interaction, and because these interactions were disrupted following cholesterol depletion by methyl‐β‐cyclodextrin (MβCD) treatment, or cholesterol sequestration by saponin. A mutant CD9 molecule lacking all palmitoylation sites was not precipitated by digitonin under conditions in which wild‐type CD9 was precipitated, indicating a role of palmitoylation for the interaction with cholesterol. Finally, upon ligation of tetraspanins on the surface of a lymphoid B cell line, the tyrosine phosphorylation of several proteins, including the vav nucleotide exchange factor, was inhibited when cells were pretreated with MβCD, and increased when they were treated with MβCD/cholesterol complexes. Thus, there is a physical and functional link between tetraspanins and cholesterol.