The photosynthetic reaction center of photosystem I (PS I) contains a phylloquinone molecule (A1) which acts as a transient electron acceptor. In PS I form the cyanobacterium Synechocystis PCC 6803 under reducing conditions, we have photoaccumulated an EPR signal assigned to the phylloquinone radical anion. The phylloquinone EPR spectrum has been studied in oriented multilayers of PS I using EPR at 9 GHz. In addition, the phyllosemiquinone spectrum has been obtained at 283 GHz using high-field, high-frequency EPR spectroscopy. From the orientation dependence of the spectrum at 9 GHz and the resolved g values obtained at 283 GHz, the phyllosemiquinone ring plane was determined to be almost perpendicular to the membrane (76 degrees) while the oxygen-oxygen (O-O) axis of the quinone was found to make an approximate 63 degrees angle to the membrane plane. The orientation of the ring plane is similar to that determined for the quinone electron acceptor (QA) in the purple bacterial reaction center, while the orientation of the O-O axis is significantly different. The new orientation information, when taken with data in the literature, allows the position of the phylloquinone in the reaction center to be better defined.