Visual phototransduction takes place in photoreceptor cells. Light absorption by rhodopsin leads to the activation of transducin as a result of the exchange of its GDP for GTP. The GTP-bound ⍺-subunit of transducin then activates phosphodiesterase (PDE), which in turn hydrolyzes cGMP leading to photoreceptor hyperpolarization. Photoreceptors return to the dark state upon inactivation of these proteins. In particular, PDE is inactivated by the protein complex R9AP/RGS9-1/Gβ5. R9AP (RGS9-1 anchor protein) is responsible for the membrane anchoring of this protein complex to photoreceptor outer segment disk membranes most likely by the combined involvement of its C-terminal hydrophobic domain as well as other types of interactions. This study thus aimed to gather information on the structure and membrane binding of the C-terminal hydrophobic segment of R9AP as well as of truncated R9AP (without its C-terminal domain, R9AP∆TM). Circular dichroism and infrared spectroscopic measurements revealed that the secondary structure of R9AP∆TM mainly includes ⍺-helical structural elements. Moreover, intrinsic fluorescence measurements of native R9AP∆TM and individual mutants lacking one tryptophan demonstrated that W79 is more buried than W173 but that they are both located in a hydrophobic environment. This method also revealed that membrane binding of R9AP∆TM does not involve regions near its tryptophan residues, while infrared spectroscopy validated its binding to lipid vesicles. Additional fluorescence measurements showed that the C-terminal segment of R9AP is membrane embedded. Maximum insertion pressure and synergy data using Langmuir monolayers suggest that interactions with specific phospholipids could be involved in the membrane binding of R9AP∆TM. Copyright © 2021 Elsevier B.V. All rights reserved.