The thromboxane A₂ receptor (TP) is a G protein-coupled receptor implicated in the regulation of pharmacological events such as platelet aggregation as well as constriction and proliferation of vascular and bronchiolar smooth muscle cells. This receptor is expressed as two alternatively spliced isoforms (TPα and TPβ) which shares the first 328 amino acids. Previous studies have demonstrated that alternative splicing of the C-tail of TP generates isoforms of the receptor showing distinct internalization characteristics. In order to identify putative proteins which are implicated in this specific regulation, we used the yeast two-hybrid system to screen for proteins that interact with the C terminus of the β isoform of the receptor (TPβ). This screen identified Receptor for Activated C-Kinase 1 (RACK1) as a new TPβ-interacting protein. Here, we show that RACK1 directly binds to the C-terminus and the first intracellular loop of TPβ. We demonstrate with co-immunoprecipitation studies in HEK293 cells that there is a constitutive RACK1-TPβ association, which is not modulated by stimulation of the receptor. We observed that cell surface expression of TPβ was promoted when RACK1 was overexpressed, while it was inhibited when endogenous RACK1 expression was knocked down by siRNA. Our data suggest that the effect of RACK1 on the export of receptors is specific to some GPCRs since inhibition of RACK1 expression also affected cell surface expression of the angiotensin II type 1 receptor and CXCR4 but not of β₂-adrenergic and prostanoid DP receptors. We also report that overexpression of RACK1 promoted, while knockdown of RACK1 by siRNA reduced, the agonist-induced internalization of TPβ. Confocal microscopy revealed that RACK1 predominantly co-localizes with TPβ at the plasma membrane in non-stimulated HEK293 cells and that agonist stimulation promoted the RACK1-TPβ co-localization in intracellular compartments. Impaired cell surface expression of TPβ in RACK1-depleted cells was also confirmed by confocal microscopy. Our data demonstrate for the first time a direct interaction between RACK1 and a GPCR and identify a novel role for RACK1 in the regulation of GPCR cell surface expression and internalization.