Akt/protein kinase B (PKB) is a serine/threonine kinase that regulates a variety of cellular responses. To provide information on the spatial and temporal dynamics of Akt/PKB activity, we have developed genetically encoded fluorescent indicators for Akt/PKB. The indicators contain two green fluorescent protein mutants, an Akt/PKB substrate domain, flexible linker sequence, and phosphorylation recognition domain. A phosphorylation of the substrate domain in the indicators caused change in the emission ratio based on fluorescent resonance energy transfer between the two green fluorescent protein mutants. To let the fluorescent indicators behave as endothelial nitric-oxide synthase and Bad, which are endogenous Akt/PKB substrates, they were fused with the Golgi target domain and mitochondria target domain, respectively. The indicators thus colocalized with the endogenous substrates conferred their susceptibilities to phosphorylation by Akt/PKB. We showed that the Golgi-localized indicator responded to the stimulation with 17beta-estradiol (E2) and insulin in endothelial cells. In addition, E2 elicited the phosphorylation of the mitochondria-localized indicator in the endothelial cells, but no phosphorylation was observed by E2 or by insulin of the diffusible indicator that has no targeting domain. The difference in the results with the three indicators suggests that the activated Akt/PKB is localized to subcellular compartments, including the Golgi apparatus and/or mitochondria, rather than diffusing in the cytosol, thereby efficiently phosphorylating its substrate proteins. E2 triggered the phosphorylation of the mitochondria-localized indicator, whereas insulin did not induce this phosphorylation, which suggests that the localization of the activated Akt/PKB to the mitochondria is directed differently between insulin and E2 via distinct mechanisms.