The adenosine A₁ receptor belongs to the family of G Protein-coupled receptors (GPCRs). Its activation causes inhibition of neurotransmitter release in neurons, inhibition of adenylyl cyclase and opening of G protein activated inwardly rectifying K⁺ (GIRK) channels in atrial myocytes, resulting in reduced heart rate and contractility. Due to its protective effect in various tissues, the A₁ receptor is considered a potential target for drug development. Because it interacts preferably with members of the G_i/o family, the receptor activation can be retraced by observing the activity of downstream targets of the G_i/o-Protein, like GIRK channels and adenylylcyclases. Its activation can be monitored with high temporal resolution using endogenous or heterologously expressed GIRK channels as target of bg subunits released from G_i/o. This however does not provide information of the agonist-induced conformational change of the receptor protein. To get direct experimental access to the ligand-induced conformational change utilizing fluorescence resonance energy transfer (FRET), yellow fluorescent protein (eYFP) was fused to the carboxy terminus and cyan fluorescent protein (eCFP) was inserted into the third cytoplasmic loop of the A₁ receptor. The ligand-induced conformational change of this A₁-FRET sensor (A₁-FS) can be monitored in real-time in intact, living cells. Moreover the kinetics of the conformational change can be compared with the kinetics of subsequent reactions and target (GIRK) activation. Therefore the A₁-FS was characterized by examination of transfected HEK293 cells, showing that it remains a functional 7TM receptor, although the added fluorophores share about two-thirds of the protein mass. Whole-cell patch clamp recordings of HEK293 cells which were co-transfected with GIRK1 and GIRK4 subunits displayed adenosine evoked GIRK currents demonstrating the ability of the A₁-FS to activate the Gi/o-coupled pathway. To determine whether the distance among CFP and YFP is in the range required for FRET measurements (2 – 8 nm), YFP was damaged by photobleaching. This resulted in an increase of CFP emission intensity of 25%. This indicates that the fluorophores are localized within a distance of 6 nm. Dynamic FRET measurements showed a concentration-dependend change in the FRET ratio upon application of adenosine with an EC₅₀ of approximately 1 mM. The half-time of the response was about 500 ms under saturating conditions (10 mM). The A₁-FS can be used to characterise different agonists and other compounds, such as allosteric enhancers.