Ligand-gated ion channels are multimeric membrane proteins with key roles within various signal transduction pathways of different cell types. A detailed knowledge of the ligand binding and activation mechanism of these channels is essential for understanding basic cellular functions and pathological processes. Information regarding the gating mechanism, single-channel conductance and selective ion permeability of ligand-gated ion channels can be gathered by means of the patch-clamp technique. The confocal patch-clamp fluorometry (cPCF) technique can additionally measure ligand binding and the triggered conformational changes within the channel protein. Unfortunately, the ligand concentration which can be used in combination with cPCF is restricted to the lower micromolar range. Herein, we propose a new approach to measure binding and gating by combining cPCF with Förster resonance energy transfer (FRET) measurements. As model system we choose retinal homotetrameric cyclic nucleotide-gated (CNGA1) channels. Binding of a cyclic nucleotide to the intracellularly-located binding domains (CNBDs) promotes an allosteric conformational change that leads to channel opening. Due to the concentration range in which this channel operates, ligand binding studies with fluorescently labelled ligands were not possible until now. To this aim, CNGA1 channels were expressed in Xenopus oocytes and their function was tested using inside-out patches. As FRET pair, the CNGA1 channel labelled at the C-terminus and a novel fluorescently-labelled cGMP derivative, 8-[DY547-P1]-AET-cGMP (P1-cGMP) were used. When the labelled ligand binds to the channel’s binding site, energy from the donor fluorophore (eGFP) is transferred to the acceptor fluorophore (DY547-P1), whose absorption spectrum overlaps with the emission spectrum of the donor. FRET efficiency was determined by measuring the decrease of donor fluorescence, allowing in this way the quantification of ligand binding. The efficiency of P1-cGMP to activate the CNGA1-eGFP channel was close to that of the physiological ligand, cGMP, 90 ± 3% (± SEM) in the presence of saturating [P1-cGMP]. The potency of the P1-cGMP to activate the CNGA1-eGFP channels was factor 10 higher in comparison with that of cGMP. The eGFP-labelling procedure had only a minor influence on the channel’s functional characteristics (EC50=27.9 µM P1-cGMP, H=2.1, n=5-8 vs. EC50=35.3 µM cGMP, H=2.6, n=5-8). The high FRET efficiency observed in combination with cPCF allowed us to study simultaneous ligand binding and channel activation. Our results showed that at concentrations lower than ~10 µM P1-cGMP, binding exceeds gating and at higher concentrations gating exceeds binding. In conclusion, the method proposed herein represents an elegant approach to measure ligand binding over a wider concentration range and to monitor binding to individually labelled subunits selectively.