Bacterial endotoxin activity is elevated in patients with decompensated chronic heart failure (HF) and acts as a potent stimulus for immune activation. We sought to determine whether endotoxin, at an activity level seen in vivo (around 0.6 EU/ml), is sufficient to stimulate the secretion of tumor necrosis factor-α (TNF-α) and TNF-α soluble receptor (sTNFR2) in ex vivo whole blood from patients with HF. We studied 15 patients with HF (aged 65 ± 1.9 years, New York Heart Association class 2.1 ± 0.3, left ventricular ejection fraction 31 ± 5%; mean ± SEM), of whom 5 had cardiac cachexia, and 7 healthy control subjects (59 ± 5 years, p = NS). Reference endotoxin was added to venous blood at concentrations of 0.6, 1.0, and 3.0 EU/ml, and was incubated for 6 hours. Endotoxin induced a dose-dependent increase in TNF-α release (p <0.05 in all groups). Patients with noncachectic HF produced significantly more TNF-α compared with controls after stimulation with 0.6, 1.0, and 3.0 EU/ml of endotoxin (113 ± 46 vs 22 ± 4 [p = 0.009], 149 ± 48 vs 34 ± 4 [p = 0.002], and 328 ± 88 vs 89 ± 16 pg/ml [p = 0.002], respectively; mean ± SEM). Patients with cardiac cachexia produced significantly less TNF-α compared with patients without cardiac cachexia for all given concentrations (all p <0.05, analysis of variance p = 0.02). Production of sTNFR2 was greater at all concentrations of endotoxin versus controls (all p <0.05, analysis of variance p = 0.002). Plasma endotoxin levels were higher in patients with cardiac cachexia (4.3 times higher than in control subjects, p <0.005). Thus, low endotoxin activity, at levels seen in vivo in patients with HF, induces significant TNF-α and sTNFR2 production ex vivo. These results suggest that elevated plasma endotoxin activity observed in patients with HF is of pathophysiologic relevance.