Cytochrome b 6 f complex with stoichiometrically bound β-carotene molecule was purified from spinach chloroplasts. The configuration of this β-carotene was studied by reversed-phase HPLC and resonance Raman spectroscopy. Both the absorption spectrum of this β-carotene in dissociated state and the Raman spectrum in native state can be unambiguously assigned to a 9-cis configuration. This finding is in contrast to the predominantly all-trans isomers commonly found in membranes and protein-pigment complexes of chloroplasts, suggesting that the 9-cis-β-carotene is an authentic component and may have a unique structural and functional role in cytochrome b 6 f complex.