Lipid A from the photosynthetic bacterium Rhodobacter sphaeroides (RSLA) has been previously shown to antagonize many of the effects of endotoxins from more pathogenic gram-negative bacteria. We have reported on the synthesis of the proposed structure of RSLA and determined that bacterially derived RSLA is not identical to its proposed structure (W.J. Christ, P. D. McGuinness, O. Asano, Y. Wang, M. A. Mullarkey, M. Perez, L. D. Hawkins, T. A. Blythe, G. R. Dubuc, and A. L. Robidoux, J. Am. Chem. Soc. 116:3637-3638, 1994). Here we report results of analyzing the antagonistic and agonistic activities of bacterially derived RSLA in comparison with the activities of chemically synthesized material of the proposed structure of RSLA and analogs. Results indicated that all compounds were approximately equally potent at inhibiting endotoxin-induced release of tumor necrosis factor alpha from human monocytes and human whole blood as well as endotoxin-induced generation of nitric oxide in murine macrophages. In addition, all compounds were of equivalent potencies at inhibiting the binding of 125I-labelled lipopolysaccharide derivatized with 2-(p-azido-salicylamido) ethyl-1-3'-dithiopropionate to murine macrophages. Higher concentrations of bacterially derived RSLA (10 to 100 microM) were agonistic in human and murine assays. In gamma interferon-treated murine macrophages, agonism was exhibited at concentrations as low as 100 nM. In contrast, all synthetic materials were either dramatically less agonistic or devoid of agonistic activity when tested at concentrations as high as 100 microM. It is possible either that bacterially derived RSLA contains a small amount of a highly agonistic impurity or that the agonistic activity of RSLA is intrinsic to its molecular structure. In either case, these biological results support our previous report concluding that biologically derived RSLA is not identical to synthetic material of its proposed structure.