•We find specific dimerization independent of cysteine residues.•ChR2 shows a structural change in helix F upon light excitation as in other retinal proteins.•We find additional structural changes in either helix B or the dimerization interface. Channelrhodopsin is a cation channel with the unique property of being activated by light. To address structural changes of the open state of the channel, two variants, which contain either 1 or 2 wild-type cysteines, were derivatised with nitroxide spin label and subjected to electron paramagnetic resonance spectroscopy. Both variants contained the C128T mutation to trap the long-lived P3520 state by illumination. Comparison of spin–spin distances in the dark state and after illumination reflect conformational changes in the conductive P3520 state involving helices B and F. Spin distance measurements reveal that channelrhodopsin forms a dimer even in the absence of intermolecular N-terminal cysteines. ChR2 and ChR2bind by electron resonance (View interaction)