Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels mediating neurotransmission in the peripheral and central nervous system as well as at the neuromuscular junction. Much is known about the gating behavior of these channel proteins, however, the reciprocal relationship between activation state and agonist binding is still elusive. It has been shown that the confocal patch-clamp fluorometry (cPCF) (Biskup et al., 2007) is a suitable technique to study activation-state dependent agonist binding by combining the electrophysiological patch-clamp technique and confocal microscopy employing fluorescently tagged agonist molecules. Herein, we adapted this technique for whole-cell recordings with HEK293 cells to study adult muscle-type nAChRs. First, we synthesized a fluorescently tagged acetylcholine derivative by linking the dye Cy3 to the acetylcholine moiety via a short polyethylene glycol linker. The resulting derivative Cy3-ACh was found to be a highly efficient (Imax,Cy3-ACh/Imax,ACh=0.97±0.01; n=3; values are means ± S.E.M.) and highly potent agonist. The apparent affinity, EC50, and the Hill coefficient, H, both derived by fitting the Hill equation to averaged relative peak currents I/Imax (n=3 to 8), were 0.99 µM and 1.8, respectively. Fast concentration jumps were realized by a piezo-driven double-barreled application pipette. Because desensitization of cells attached to the chamber bottom was slowed in comparison to the desensitization of cells lifted up after whole-cell configuration was established (means±S.E.M.: 186±21.3 ms; n=5 vs. 85.4±12.8 ms; n=5; p<0.05 t-test), we concluded that solution exchange is faster and more defined in lifted cells. Confocal fluorescence imaging was realized with a confocal laser scanning microscope (LSM710, Zeiss, Germany). Cy3-ACh was excited with a 543 nm HeNe laser line. To define the position of the lifted cell for confocal imaging, the background was stained with a reference dye, Dy647, excited with a 633 nm HeNe laser line. We confirmed that Dy647 did neither bind to the nAChRs nor to the plasma membrane. Regarding unspecific fluorescence signals due to the patch pipette, we found unpolished borosilicate glass preferable compared to quartz glass: After application of 10 µM Cy3-ACh, unspecific fluorescence of quartz pipettes was more than 3 times higher compared to borosilicate pipettes (4.9±1.3 a.u. in quartz; n=4 vs. 1.3±0.3 a.u. in borosilicate; n=3; p<0.1 t-test). After application of Cy3-ACh to nAChR-expressing HEK293 cells a clear membrane staining was visible, which was not found in non-expressing control cells. The binding was reversible and the agonist could be washed off completely after 13 seconds. Repeated application of Cy3-ACh to the same cell resulted in similar fluorescence intensities. Herein, we showed that cPCF can be applied to directly relate agonist binding to different activation states of nAChRs. It can be concluded that confocal PCF is a flexible approach to study ion channels and ionotropic receptors not only in excised membrane patches but also in whole mammalian culture cells, expanding the scope of application significantly.