For the first time to our knowledge, X-ray absorption spectroscopy (XAS) has been used to investigate the environment of putative Zn2+ binding sites in rhodopsin. We studied native purified nondeionized rhodopsin without any further addition of Zn2+, as well as with 1.5 mol of Zn 2+ - as zinc chloride - per mole of protein. Three different binding sites in rhodopsin were considered based on computational chemistry studies, and a quantitative analysis of the XAS signal was performed by fitting the experimental data to their simulated XAS spectra. Our results demonstrate that Zn2+ is intrinsically bound to rhodopsin and are compatible with the existence of an octahedral coordination involving six oxygen atoms in the first shell (average Zn-O distance of 2.08 Å), and with a second coordination shell containing one or two phosphorus or sulfur atoms at an average distance of 2.81 Å. © 2009 The Authors.