The primary donor in single crystals of photosystem I (PS 1) obtained from the thermophilic cyanobacterium Synechococcus elongatus is investigated by EPR and ENDOR techniques. The cation radical P-700(+.) was generated chemically or photolytically in the single crystals, and angular dependent spectra were obtained at a temperature of 100 K in the frozen state. The principal values and corresponding axes orientations were determined for three different proton hyperfine coupling (hfc) tensors. On the basis of the tensor magnitudes, their symmetry, and the relative orientations of their axes, they were assigned to the protons of three methyl groups of a single chlorophyll (Chl) alpha molecule. From X-ray crystallography of PS 1 single crystals, it is known that P-700 is structurally a chlorophyll dimer (Klukas et al., J. Biol. Chem. 1999, 274, 7361). The EPR/ENDOR experiments show that the second chlorophyll half carries no or very little (less than or equal to 15%) spin density. The molecular plane of the spin-carrying Chl alpha molecule in P-700(+.) is parallel to the crystallographic c axis. From the orientation of the hf tensors of the methyl groups with respect to this axis, the orientation of the Chl alpha molecule could be determined. The orientation of this Chl alpha is very similar to that of the two symmetrically placed bacteriochlorophyll molecules forming the primary donors P-865 and P-960 in the RC of the purple bacteria Rhodobacter sphaeroides and Rhodopseudomonas viridis, respectively. These results indicate a structural similarity of the electron donors in purple bacteria and photosystem 1 and point to a common evolutionary origin of both types of reaction centers.