Although Interleukin-6 (IL-6) plays an important role in the pathophysiology of traumahemorrhage and resuscitation, the cellular origin of this inflammatory cytokine remains unknown. This study was undertaken to determine whether Kupffer cells (KC) are a major source of IL-6 release following traumahemorrhage and resuscitation. KC numbers were significantly (p > .05) reduced in vivo with gadolinium chloride (GdCI3; 10 mg/kg IV). KC-reduced (KC(−)) and KC-normal (saline-treated; KC(+)) rats underwent laparotomy (i.e., trauma-induced), followed by either sham operation or hemorrhage. Hemorrhaged rats were bled to and maintained at a mean arterial pressure of 40 mmHg until 40% of the shed blood volume was returned as Ringer's lactate, and then resuscitated with Ringer's lactate (four times shed blood volume over 1 h). Results indicate that KC reduction per se had no effect on any measured parameter at any time. At 0.5 and 2.0 h postresuscitation, mean arterial pressure, heart rate, cardiac output, stroke volumen, and hematocrit were reduced to a similar extent in both the KC(+) and KC(−) hemorrhage groups. KC reduction did, however, significantly reduce plasma IL-6 concentration (means ± S.E.; U/ml) at both 0.5 h (KC(+) = 709 ± 391 vs. KC(−) = 159 ± 5) and at 2.0 h (KC(+) = 527 ± 394 vs. KC(−) = 83 ± 20) postresuscitation. In conclusion, this study demonstrates that KC are a major source of in vivo IL-6 release following trauma-hemorrhage and resuscitation.