Tetraspanins CD9 and CD151 have been implicated in cellular motility and intercellular adhesion in several cellular types. Here, we have studied the subcellular localization and the functional role of these molecules in primary melanocytes. We found that endogenous tetraspanins preferentially clustered in areas of melanocyte homotypic intercellular contacts and at the tips of dendrites. These observations were further confirmed using time-lapse fluorescence confocal microscopy of melanocytes transfected with CD9- and CD151-GFP (green fluorescent protein) constructs, suggesting an involvement of these proteins in cellular contacts and migration. Cell adhesion and migration assays performed using blocking monoclonal antibodies against CD9 and CD151 showed no significant effect on cell-extracellular matrix adhesion, whereas the migration of melanocytes was significantly enhanced. The regulation of the migratory capacity of melanocytes by CD9 and CD151 was further confirmed knocking down the endogenous expression of these tetraspanins with small interference RNA oligonucleotides. Therefore, tetraspanin molecules are localized at motile structures in primary human melanocytes regulating the migratory capacity of these cells.