This review summarizes key aspects of tetraspanin proteins, with a focus on the functional relevance and structural features of these proteins and how they are organized into a novel type of membrane microdomain. Despite the size of the tetraspanin family and their abundance and wide distribution over many cell types, most have not been studied. However, from studies of prototype tetraspanins, information regarding functions, cell biology, and structural organization has begun to emerge. Genetic evidence points to critical roles for tetraspanins on oocytes during fertilization, in fungi during leaf invasion, in Drosophila embryos during neuromuscular synapse formation, during T and B lymphocyte activation, in brain function, and in retinal degeneration. From structure and mutagenesis studies, we are beginning to understand functional subregions within tetraspanins, as well as the levels of connections among tetraspanins and their many associated proteins. Tetraspanin-enriched microdomains (TEMs) are emerging as entities physically and functionally distinct from lipid rafts. These microdomains now provide a context in which to evaluate tetraspanins in the regulation of growth factor signaling and in the modulation of integrin-mediated post-cell adhesion events. Finally, the enrichment of tetraspanins within secreted vesicles called exosomes, coupled with hints that tetraspanins may regulate vesicle fusion and/or fission, suggests exciting new directions for future research.