The FSH receptor (FSH-R) is a member of the rhodopsin-like subfamily of G protein-coupled receptors that undergoes homologous desensitization upon agonist stimulation. In immortalized cell lines overexpressing the FSH-R, G protein-coupled receptor kinases (GRKs) and β-arrestins are involved in the phosphorylation, uncoupling, and internalization of this receptor. In an effort to appreciate the physiological relevance of GRK/β-arrestin actions in natural FSH-R-bearing cells, we used primary rat Sertoli cells as a model. GRK2, -3, -5, -6a, and -6b and β-arrestins 1 and 2 were expressed in primary rat Sertoli cells. Overexpression of these different GRKs and β-arrestins in primary rat Sertoli cells significantly attenuated the FSH-induced cAMP response, and FSH rapidly triggered a relocalization of endogenously expressed GRK2, -3, -5, and -6 and β-arrestins 1 and 2 from the cytosol to the membranes. These results highlight the relationship existing between the GRK/β-arrestin regulatory system and the FSH-R signaling machinery in a physiological model.