We have investigated the functional coupling of α and β isoforms of the human thromboxane A 2 receptor (TP) to Gα 16 and Gα 12 members of the G q and G 12 families of heterotrimeric G proteins in human embryonic kidney (HEK) 293 cell lines HEK.α10 or HEK.β3, stably over-expressing TPα and TPβ, respectively. Moreover, using HEK.TP Δ328 cells which over-express a variant of TP truncated at the point of divergence of TPα and TPβ, we investigated the requirement of the C-tail per se in mediating G protein coupling and effector activation. Both TPα and TPβ couple similarly to Gα 16 to affect increases in inositol 1,4,5-trisphosphate (IP 3) and mobilisation of intracellular calcium ([Ca 2+] i) in response to the TP agonist U46619. Whilst both TP isoforms mediated [Ca 2+] i mobilisation in cells co-transfected with Gα 12, neither receptor generated corresponding increases in IP 3, indicating that the Gα 12-mediated increases in [Ca 2+] i do not involve PLC activation. Verapamil, an inhibitor of voltage dependent Ca 2+ channels, reduced [Ca 2+] i mobilisation in TPα and TPβ cells co-transfected with Gα 12 to approximately 40% of that mobilised in its absence, whereas [8-( N,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate, hydrochloride] (TMB-8), an antagonist of intracellular Ca 2+ release, had no effect on [Ca 2+] i mobilisation by either receptor isoform co-transfected with Gα 12. Despite the lack of differential coupling specificity by TPα and TPβ, TP Δ328 signalled more efficiently in the absence of a co-transfected G protein compared to the wild type receptors but, on the other hand, displayed an impaired ability to couple to co-transfected Gα 11, Gα 12 or Gα 16 subunits. In studies investigating the role of the C-tail in influencing coupling to the effector adenylyl cyclase, similar to TPα but not TPβ, TP Δ328 coupled to Gα s, leading to increased adenosine 3′,5′-cyclic monophosphate (cAMP), rather than to Gα i. Whereas TP Δ328 signalled more efficiently in the absence of co-transfected G protein compared to the wild type TPα, co-transfection of Gα s did not augment cAMP generation by TP Δ328. Hence, from these studies involving the wild type TPα, TPβ and TP Δ328, we conclude that the C-tail sequences of TP are not a major determinant of G protein coupling specificity to Gα 11 and Gα 16 members of the G q family or to Gα 12; it may play a role in determining G s versus G i coupling and may act as a determinant of coupling efficiency.