Protein kinase C (PKC) is correlated with cell signaling pathways and also receives attention as a therapeutic target for cancer and Alzheimer-type dementia. The application of Förster/fluorescence resonance energy transfer (FRET) phenomena to detect binding between proteins and small molecules, for example, PKC and its ligands, underlies a fluorescence-based assay method suitable for high-throughput screening. To accelerate studies on PKC functions in processing signals using small molecules and the development of drugs that target PKC, novel methods for the assessment of the PKC binding affinity of compounds are necessary. We previously developed solvatochromic fluorophore-based methods for that assessment. In this study, a novel method for a FRET-based PKC binding assay was developed and is expected to overcome the limitations of solvatochromic fluorophores.Key words protein kinase C; protein kinase C ligand; ligand screening; Förster resonance energy transfer; fluorescence resonance energy transfer Förster/fluorescence resonance energy transfer (FRET) is the transfer of excitation energy from a fluorescence donor to an acceptor. FRET efficiency and sensitivity depend on the distance between the donor and the acceptor. The FRET phenomenon can be applied as a spectroscopic measure and a probe of conformational change of macro-biomolecules [1][2][3] and a FRET-mediated competitive assay can be used for studies of the binding between two molecules. 4,5) Radioisotopebased methods are extremely sensitive and are widely used for ligand binding assays, but fluorescent-based assays, which are suitable for high-throughput screening are free of hazards associated with radioactivity. Previously, we described fluorescence-based ligand screening assays as an alternative to radioisotope assays. 6,7)Protein kinase C (PKC) isozymes, which are classified as Ser/Thr kinases, play a critical role in cellular signaling pathways related to proliferation, 8,9) differentiation, 10) and apoptosis.11,12) PKC has 11 isozymes which are classified into three subtypes: conventional PKC (cPKC; α, β I/II , γ), novel PKC (nPKC; δ, ε, η, τ) and atypical PKC (aPKC; ζ, λ, ι). The binding of diacylglycerols (DAG) to the C1 domain of PKC is an important step in an activation process except in the case of aPKC which contains an atypical C1 domain which fails to bind to DAG.13) Various synthetic PKC ligands targeting the C1 domain have been developed as chemical probes or drug candidates by radioisotope assays.14-17) We have previously developed a fluorescent assay for PKC-ligand binding affinity using a synthetic PKC δC1b domain labeled with a solvatochromic fluorophore on the edge of its ligand-binding pocket. This can detect ligand binding through changes in the surrounding environment.18) However, this method would not be suitable for high-throughput screening. The reason might be due to several possibilities: For instance, the change of fluorescence intensity is affected by properties of test compounds. It would be concerned that a fluorescen...