Chronic agonist stimulation of the β2 Adrenergic Receptors (β2ARs) leads to their lysosomal trafficking and degradation. Previous studies demonstrated that agonist-induced β2AR ubiquitination is necessary for lysosomal targeting and degradation of the receptor. We have now found that the de-ubiquitinating enzymes USP33 and USP20 are recruited to the β2AR complexes, by using cellular co-immunoprecipitation assays. This led to our hypothesis that USP33 and USP20 could function to reverse β2AR ubiquitination and thus, could be involved in the regulation of receptor trafficking. Indeed, coexpression of the β2AR with USP33 or USP20 (USP33/20) results in a dramatic reduction of isoproterenol-stimulated ubiquitination of the receptor. In contrast, similar coexpression of catalytically inactive USP33/20 mutants, which retains receptor binding, does not lead to any decrease in receptor ubiquitination. When HEK-293 cells expressing the β2AR are exposed to 10 μm isoproterenol for 6h, internalized β2ARs are found to colocalize with the lysosomal marker protein, LAMP2. Wild type USP33/20, but not the catalytically inactive mutants expression abolishes this colocalization. Moreover, in the presence of USP33/20, β2ARs are found to be redirected to the plasma membrane even in the continued presence of agonist thus inhibiting lysosomal trafficking while concomitantly promoting receptor recycling from the late endosomal compartments as well as resensitization of recycled receptors at the cell surface. Inhibition of both USP20 and USP33 expression by SiRNA prevents completely the recycling and resensitization of the receptor whereas inhibition of only one of the two enzymes does not. Finally, dissociation of constitutively bound USP20 and USP33 from the β2-AR immediately after agonist-stimulation and re-association upon prolonged agonist treatment allows receptors to first become ubiquitinated and then deubiquitinated thus providing a “trip switch” between degradative and recycling pathways at the late endosomal compartments. In conclusion, our results suggest that by de-ubiquitinating the internalized β2ARs, USP20 and USP33 prevent their lysosomal trafficking and promotes receptor recycling and resensitization to the plasma membrane. USP20 and USP33 thus serve as novel regulators that dictate both post-endocytic sorting as well as the intensity and extent of β2-AR signalling from the cell surface.ββ