The mainstay of assessing guanosine diphosphate release by the alpha-subunit of a heterotrimeric G-protein is the [(35)S]guanosine 5′-O-(3-thiotriphosphate) (GTPgammaS) radionucleotide-binding assay. This assay requires separation of protein-bound GTPgammaS from free GTPgammaS at multiple time points followed by quantification via liquid scintillation. The arduous nature of this assay makes it difficult to quickly characterize multiple mutants, determine the effects of individual variables (e.g., temperature and Mg(2+) concentration) on nucleotide exchange, or screen for small molecule modulators of Galpha nucleotide binding/cycling properties. Here, we describe a robust, homogeneous, fluorescence polarization assay using a red-shifted fluorescent GTPgammaS probe that can rapidly determine the rate of GTPgammaS binding by Galpha subunits. See PMID: 20662737.