We have characterized a xanthophyll binding site, called V1, in the major light harvesting complex of photosystem II, distinct from the three tightly binding sites previously described as L1, L2, and N1. Xanthophyll binding to the V1 site can be preserved upon solubilization of the chloroplast membranes with the mild detergent dodecyl-alpha-d-maltoside, while an IEF purification step completely removes the ligand. Surprisingly, spectroscopic analysis showed that when bound in this site, xanthophylls are unable to transfer absorbed light energy to chlorophyll a. Pigments bound to sites L1, L2, and N1, in contrast, readily transfer energy to chlorophyll a. This result suggests that this binding site is not directly involved in light harvesting function. When violaxanthin, which in normal conditions is the main carotenoid in this site, is depleted by the de-epoxidation in strong light, the site binds other xanthophyll species, including newly synthesized zeaxanthin, which does not induce detectable changes in the properties of the complex. It is proposed that this xanthophyll binding site represents a reservoir of readily available violaxanthin for the operation of the xanthophyll cycle in excess light conditions.