Lipopolysaccharide (LPS) prepared from Enterobacter agglomerans was tested for its ability to stimulate mediator release in vitro and in vivo, together with its ability to induce pulmonary inflammatory reactions. As observed with LPS prepared from E. coli and S. minnesota (1,2), E. agglomerans LPS produced both interstitial pulmonary inflammation and febrile responses following inhalation of 50 ug per day per rabbit for three consecutive days. Interestingly, the febrile responses observed on days two and three were notably less than those observed on day one, suggesting the induction of "tolerance" to LPS challenge. Interleukin 1 (IL-1), which we have shown to be closely similar to if not identical with rabbit alveolar macrophage-derived endogenous pyrogen (3), was detected in lavage fluid obtained after the third aerosol challenge, but not after a single aerosol challenge. However, alveolar cells harvested from all animals given a single LPS challenge showed marked spontaneous IL-1 secretion in vitro, whereas cells from animals challenged with LPS aerosol for three days showed little, again suggesting some degree of LPS tolerance after three days. When further challenged with E. agglomerans LPS in vitro, however, AM from all experimental and phosphate-buffered saline (PBS) control animals showed TL-1 production. Similar results were obtained when production of a fibroblast growth factor was measured. Lavage fluids from animals challenged with LPS aerosols for three days contained more growth factor than did that from animals challenged for only one day, whereas alveolar cells from one-day animals showed considerably more spontaneous secretion of growth factor in vitro than did those from three-day animals. A cytotoxic factor for L-929 fibroblast cultures was detected in lavage fluids from LPS aerosol-challenged rabbits obtained following either one or three days of LPS inhalation, although little if any spontaneous secretion by alveolar cells was observed in vitro. E. agglomerans LPS readily stimulated alveolar cells from all animals to secrete this cytotoxic factor in vitro, however. Alveolar cells harvested following a single or multiple challenges with LPS or PBS showed little if any spontaneous production of superoxide anion. However, alveolar cells from LPS challenged (but not PBS-challenged rabbits readily secreted superoxide when stimulated in vitro with phorbol myristate acetate (PMA). The studies clearly demonstrate that inhaled E. agglomerans LPS induces pulmonary inflammation and fever, in association with release of IL-1, fibroblast growth factors and cytotoxic factors from alveolar cells.