The reaction center complex of heliobacteria contains three kinds of chlorophyll pigments, bacteriochlorophyll g_F (BChl g_F), its 13²-epimer BChl g_F′ and 8¹-hydroxy-chlorophyll a_F (8¹-OH-Chl a_F). Because the full stereochemistry of these naturally occurring chlorophyllous pigments has remained unknown, we determined the stereochemistry of both BChl g_F and 8¹-OH-Chl a_F extracted from Heliobacterium modesticaldum. The configurations of the specific functional groups at ring-B as well as those at ring-D and -E were investigated by use of nuclear Overhauser effect correlations in their ¹H-NMR spectra and circular dichroism spectra, as well as by modified Mosher's method in their chemical modification: (1) E-configuration was confirmed for the 8-ethylidene group at ring-B in BChl g_F, (2) R-configuration was identified for the 1-hydroxyethyl group at ring-B in 8¹-OH-Chl a_F and (3) 13²-(R)-, 17-(S)- and 18-(S)-configurations at ring-D and -E in both BChl g_F and 8¹-OH-Chl a_F were confirmed. These stereochemistries enabled us to discuss their biosynthesis and to propose possible routes for preparation of ethylidene and 1-hydroxyethyl groups at the 8-position.