Repetitive low-dose endotoxin, at a dose which will result in endotoxin tolerance, produces a marked but transient 2-to 3-day increase in plasma fibronectin. This elevation of fibronectin appears to contribute to increased hepatic Kupffer cell phagocytic function observed with repetitive low-dose endotoxin administration. Although numerous cell types synthesize fibronectin, hepatocytes are believed to be the major cell source of fibronectin in the plasma. Since Kupffer cells also synthesize fibronectin, we sought to determine the relative contribution of hepatic Kupffer cells, as compared to parenchymal cells, to the elevation of plasma fibronectin following repetitive low-dose endotoxin administration. Kupffer cells isolated from rats previously treated for 3 consecutive days with 100 pg Salmonella enteritidis endotoxin released greater (p < 0.01) amounts of fibronectin over time in culture (3,6,12 and 24 hr) as compared to Kupffer cells isolated from normal rats. Experiments in which fibronectin was normalized to DNA content of the cells in culture also showed similar results for fibronectin release by Kupffer cells (normal: 2.9 f 0.5 ng per pg DNA per 24 hr; endotoxin-treated: 53.3 f 1.3 ng per pg DNA per 24 hr). Hepatocytes from endotoxin-treated rats released less (p < 0.01) fibronectin over time than hepatocytes isolated from normal animals. As with Kupffer cells, results for fibronectin release by hepatocytes were similar when normalized to the DNA content (normal: 190.0 f 9.4 ng per pg DNA per 24 hr; endotoxin-treated 83.3 f 4.2 ng per pg DNA per 24 hr). However, cultured hepatocytes did synthesize and release more fibronectin than cultured Kupffer cells whether they were isolated from normal or endotoxin-treated rats. These results suggest that Kupffer cells may contribute a greater portion of the plasma fibronectin pool following endotoxin treatment than in normal animals. Furthermore, the increased release of fibronectin by Kupffer cells observed after endotoxin is coupled with decreased hepatocyte release of fibronectin.