The definitive version is available at http://www.blackwell-synergy.com/Different pigment extraction procedures and HPLC methods were tested to investigate the geometric configuration of the β-carotene in two forms of the photosystem II reaction center (Dl-D2-cytochrome (Cyt) b559) complex containing one and two β-carotene molecules per two pheophytin a. All the handling steps and HPLC analyses were done in darkness at room temperature and at 4°C. Two different pigment extracts were analyzed, a mixture of chlorophyll a, pheophytin a and 3-carotene, and the isolated p-carotene from that mixture. In both cases only the all-frans-β-carotene was detected. The chromatographic profiles were similar at both temperatures only differing in the retention times that were longer at 4°C. This result was independent of the concentration of photosynthetic starting material. Furthermore, no differences were observed between Dl-D2-Cyt b559 complexes with one and two p-carotene molecules per reaction center. The analysis of the β-carotene chromatographic peak indicated no 15-cis to all-fraws isom-erization occurred during the HPLC chromatography in our experimental conditions. Resonance Raman spectra were also recorded in the isolated Dl-D2-Cyt b559 complex at room and liquid nitrogen temperature with excitation at 514.5 nm from an Ar+ laser. Spectra of control preparations showed main bands at 1532, 1264, 1213, 1185, 1154 and 1003 cm−1 corresponding to the M-trans isomer and confirm previous results. The presence in the reaction center suspension of artificial electron acceptors such as silicomolybdate or 2,5-dibromo-3-methyl-6-iso-propyl-p-benzoquinone that are able to quench 3P680 did not modify the resonance Raman spectra of the native Dl-D2-Cyt b559 complex. The results suggest that no isomerization takes place during the laser irradiation.This work was supported by the Dirección General de Investigación Científica y Técnica (Grant PB95-0219) to R.P.Peer reviewed