Endotoxic lipopolysaccharides (LPS) isolated from Serratia marcescens, Veillonella alcalescens, and Salmonella typhosa were potent in their ability to induce fixation of complement (C') in normal guinea pig, rabbit, mouse, and human serum. The C'-fixing ability of LPS was pronounced even when assays were performed in undiluted serum, and was lost after each of four chemical modifications which resulted in loss of biological toxicities. The detoxification procedures had in common the cleavage of ester-bound, long-chain carboxylic acids. The ability of biologically active LPS to fix C' in normal guinea pig serum was reflected chiefly in dramatic uptake of classical C'3 (C'3t); fixation of C'1, C'4, and C'2 was virtually undetectable. Hence, it was the capacity for fixation of C'3t which was lost most overtly during detoxification. Addition of immune serum to the assay mixtures resulted in detectable fixation of C'1 and C'4. Biologically active LPS also fixed more of these components than did detoxified LPS. Immune serum restored the ability of detoxified LPS to fix C'3t, but whether this is by the original pathway is not yet clear. We concluded that the loss of certain biological activities and the loss of ability to fix C'3t in normal serum after LPS detoxification involved loss or rearrangement of substrates on LPS which either initiated or supported, or both, its interaction with the complement system. It was apparent that the ability to fix C' can serve as a valuable in vitro indicator of the integrity of the toxic conformation of biologically active LPS membrane fragments. These experiments supported the hypothesis that certain of the biological activities induced by endotoxins are mediated via the complement system.