A number of Eubacteria contain a photoactive yellow protein which has a photosensory function in negative phototaxis. It has been proposed that the cofactor responsible for the intense yellow color of this protein is retinal [McRee, D. E., et al. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 6533-6537]. This would make it the first eubacterial rhodopsin. Here we report the chemical structure of this chromophoric group to be p-coumaric acid, which is covalently bound to a unique cysteine in the apoprotein via a thiol ester bond, and thus not retinal. This makes PYP the first example of a protein containing p-coumaric acid, a metabolite previously found only in plants, as a prosthetic group and establishes the photoactive yellow proteins as a new type of photochemically active receptor molecule.