Nicotinic acetylcholine receptors (nAChRs) are expressed in different neuronal and non-neuronal cell types throughout the body, where they mediate fast synaptic cholinergic transmission. nAChR current responses are characterized by a rapid activation upon agonist binding followed by a complex desensitization in presence of the agonist. To contribute to the understanding of the reciprocal relationship between agonist binding and desensitization of the receptor, we applied electrophysiological as well as fluorescence techniques employing a fluorescently tagged ACh derivative, Cy3-ACh. Herein, we studied adult muscle-type nAChRs, composed of α1, β1, δ, and ε subunits in a ratio of 2:1:1:1, heterologously expressed in HEK293 cells. For electrophysiological recordings as well as for fluorescence imaging, whole cells were lifted after obtaining the whole-cell configuration and positioned in front of a double-barreled application pipette. Fast solution exchanges were realized by a piezo device. Fluorescence recordings were performed with the confocal laser scanning microscope LSM710 (Zeiss, Jena, Germany). Cy3-ACh was excited with a 543nm HeNe laser line. Values are means ± S.E.M. First, we characterized the functionality of Cy3-ACh on muscle-type nAChRs. We found that Cy3-ACh is as efficient as untagged ACh to open the channel (Imax,Cy3-ACh/Imax,ACh=0.97±0.01; n=3). The potency for Cy3-ACh (EC50= 0.91 µM) was slightly higher than for untagged ACh (EC50=2.4 µM). EC50 values were derived by fitting the Hill equation to averaged relative peak currents (I/Imax; n=5 for ACh, n=3 to 8 for Cy3-ACh). The desensitization kinetics was similar for both agonists over the whole concentration range tested (0.3 to 1000 µM). In imaging experiments, application of Cy3-ACh led to fluorescence signals, which were not observed when applied to non-transfected control cells. Agonist binding and unbinding appeared as bi-exponential time courses, whereby in both cases the first phase was too fast to be resolved. Interestingly, the second phase of fluorescence increase during agonist application was similar to the time course of desensitization. The second phase of fluorescence decrease during wash-off phase was similar to the time course of recovery from desensitization. These results led us to the following conclusions: (1) Cy3-ACh is a suitable agonist to monitor binding and unbinding in adult muscle-type nAChRs as well as their respective consequences for channel gating, (2) Our direct macroscopic approach confirmed that desensitization leads to higher agonist affinity as suggested with single-channel approaches before (Akk and Auerbach, 1996. Biophys. J.70,2652-8). (3) Recovery from desensitization is rate-limited by ligand unbinding. Hereby, we conclude that Cy3-Ach derivative can be a potential tool to study relation between ligand binding and channel gating.