Palmitoylation is a reversible and dynamic lipid modification whereby palmitic acid is added via a thioester linkage to carboxyl (C)-terminal cysteine residues. It is a prevalent feature amongst G protein coupled receptors, with up to 80% of GPCRs estimated to contain at least one cysteine within their C-tail domain that may be potentially palmitoylated. We sought to establish whether the TPα and/or TPβ isoforms of the human prostanoid thromboxane (TX) A₂ receptor (TP) are palmitoylated and to assess the functional consequences thereof. Our studies confirm that consistent with the presence of three cysteines within its unique C-tail TPβ, but not TPα, is palmitoylated. Furthermore, site directed mutagenesis and in vivo metabolic labeling studies confirmed that TPβ is palmitoylated at Cys³⁴⁷ and, to a lesser extent, at Cys^373/377. Whilst impairment of palmitoylation did not affect ligand affinity of TPβ, agonist-induced [Ca²⁺]_i mobilization was significantly reduced relative to the wild type TPβ when TPβ^C347, but not TPβ^C373 or TPβ^C373,377, was mutated suggesting that palmitoylation at Cys³⁴⁷ is specifically required for efficient Gq/phospholipase Cβ effector coupling. As TPβ, but not TPα, is reported to undergo both agonist-induced and temperature-dependent tonic internalization, albeit through distinct mechanisms, we sought to determine whether palmitoylation affects TPβ internalization. It was found that palmitoylation at Cys^373,377 is critical for TPβ internalization as TPβ^C373S, TPβ^C373,377S and TPβ^{C347,373,377S} underwent significantly reduced agonist-induced and temperature-dependent tonic internalization compared to wild type TPβ. On the other hand, whilst TPβ^C347S underwent reduced agonist-induced internalization, it underwent tonic- internalization to a similar extent as TPβ. The deficiency in agonist-induced internalization by TPβ^C347S, but not by TPβ^C373,377S nor TPβ^{C347,373,377S}, was overcome by over-expression of either β-arrestin1 or β-arrestin2. Taken together, data herein suggest that whilst palmitoylation of TPβ at Cys^373,377 is critical for both agonist- and tonic-induced internalization, palmitoylation at Cys³⁴⁷ has a role in determining which pathway is followed, be it by the β-arrestin-dependent agonist-induced pathway or by the β-arrestin-independent tonic internalization pathway.