Lipopolysaccharide (LPS) chemically extracted from gram-negative bacteria is often used in animal models to study endotoxemia. Laser confocal microscopy and immunofluorescence staining for comparison of injections of live Escherichia coli O111:B4 bacteria with LPS extracted from the same strain showed that cellular localization and time course in rat organs were markedly different after the two injections. Fluorescent staining and image analysis software allowed quantitative comparison of LPS within tissues at different times and doses. Antigenic LPS was detected in all tissues 1 hour after injection of both bacteria and LPS and was present in liver and spleen over the 28-day study period. Whole bacteria were identified in tissue macrophages for the first 48 h after injection; later, bacterial cell walls were replaced by diffuse antigenic material throughout the cytoplasm. Antigenic LPS was localized within hepatocytes only after injection of chemically purified LPS. Cellular localization of LPS in tissues is dependent on the form injected. Animal models that use purified LPS may not be representative of gram-negative bacteremia.