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Production of antisera able to recognize individual heterotrimeric G protein alpha subunits resulted in rapid expansion of information on their distribution and function. However, no antibodies that specifically recognize the active state have been available. Four-way primary screening of 763 hybridomas generated from mice immunized with guanosine 5'-O-(3-thio)triphosphate-loaded G alpha(i1) and isolated using an automated robotic colony picker identified three antibodies that interacted with the constitutively active, Q(204)L, mutant but neither the constitutively inactive, G(203)A, mutant nor wild-type G alpha(i1). This profile extended to other closely related G(i) family G proteins but not to the less closely related G alpha(s) and G alpha(q)/G alpha(11) families. Each antibody was, however, also able to identify wild-type, GDP-bound G(i) family G proteins in the presence of fluoroaluminate, which mimics the presence of the terminal phosphate of GTP and hence generates an active/transition state conformation. Stimulation of cells coexpressing a wild-type G alpha(i) subunit and the dopamine D2 receptor with the agonist ligand nor-apomorphine also allowed these conformationally selective antibodies to bind the G protein. Such reagents allow the specific identification of activated G proteins in a native environment and may allow the development of label-free screening assays for G protein-coupled receptor-mediated activation of G(i) family G proteins.