A series of substituted bacteriochlorophyll molecules, all used in reconstitution experiments of reaction centers of Rhodobacter sphaeroides (Struck et al. Biochim. Biophys. Acta 1991, 1060, 262-270), were characterized by EPR, electron-nuclear double (ENDOR), and electron-nuclear-nuclear triple (TRIPLE) resonance spectroscopy in their monomeric radical cation states. Effects of different substituents at position 3 in the porphyrin macrocycle were considered, especially for two "crosslinks" between plant and bacterial chlorophylls. These are 3-vinylbacteriochlorophyll where the "bacteria" acetyl group at position 3 was substituted by vinyl and 3-acetylchlorophyll where the "plant" vinyl group was substituted by acetyl. In addition, effects of substitutions at position 13(2) were studied. All major hyperfine coupling constants of proton and nitrogen nuclei were elucidated from the spectra and assigned to molecular positions by comparison with the parent radicals. The data were compared with those calculated by an INDO-type program, showing that INDO essentially models the effect of the different substituents correctly.