The spin-polarized radical pair of the primary donor chlorophyll P700(+) and the secondary acceptor Q(-) was studied by the electron spin echo envelope modulation (ESEEM) technique in the spinach photosystem I reaction center in which artificial quinones and fluorenones were reconstituted as Q. From the dipole interaction between the spins on P700(+) and Q(-), the distance between P700 and anilinochloronaphthoquinone or hydroxyanthraquinone was estimated to be 25.6 +/- 0.3 Angstrom or 25.7 +/- 0.3 Angstrom, respectively, suggesting their proper binding at the original phylloquinone binding site. Distances of 26.0 Angstrom with larger heterogeneity were estimated for (NO2)(3)- and (NO2)(4)-fluorenone compounds that have only one carbonyl group, suggesting their multiple orientations in the binding pocket. The lifetimes of the radical pairs estimated by the time dependency of the two-pulse ESE signals were almost temperature independent (30-80 mu s at 4-80 K), In the three-pulse ESE measurement, the lifetimes were lengthened by 10-10(3) times and showed strong temperature dependencies.