Production of antisera able to recognize individual heterotrimeric G protein 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_i1 and isolated using an automated robotic colony picker identified three antibodies that interacted with the constitutively active, Q²⁰⁴L, mutant but neither the constitutively inactive, G²⁰³A, mutant nor wild-type G_i1. This profile extended to other closely related G_i family G proteins but not to the less closely related G_s and G_q/G₁₁ 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_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.—Lane, J. R., Henderson, D., Powney, B., Wise, A., Rees, S., Daniels, D., Plumpton, C., Kinghorn, I., Milligan, G. Antibodies that identify only the active conformation of G_i family G protein subunits.