The activation of guinea pig peritoneal macrophages by Salmonella typhimurium lipopolysaccharide (LPS) was studied by using 14C-glucosamine uptake. Peritoneal exudate cells incorporated significant amounts of 14C-glucosamine when stimulated with LPS but neither purified macrophages nor nonadherent lymphocytes by themselves incorporated glucosamine. The activation of macrophages could be restored by adding nonadherent peritoneal lymphocytes, spleen cells, and lymph node cells but not thymocytes. Removal of B lymphocytes abolished the restorative capacity from active lymphoid cell populations. In contrast, B lymphocytes would restore glucosamine incorporation by macrophages stimulated with LPS but T lymphocytes did not. In addition, cell-free supernatants from LPS stimulated B lymphocytes but not from T lymphocytes could restore glucosamine incorporation by macrophages. These experiments demonstrate that LPS does not directly activate macrophages as measured by glucosamine incorporation but stimulates B lymphocytes which in turn activate macrophages.