CD8 knockout mice that are depleted of naturla killer cells by treatment with anti-asialoGM1 (CD8KO/AsGM1 mice) are resistant to injury caused by cecal ligation and puncture (CLP). However, CLP-induced injury is complex. Potential mechanisms of injury include systemic infection, cecal ischemia and translocation of bacterial toxins. Currently, it is unclear which of these modes of injury contribute to mortality in wild type mice and whether CD8KO/AsGM1 mice are resistant to any particular mechanism of injury. In the present study, we hypothesized that bacterial dissemination is the major cause of injury following CLP in wild type mice and that CD8KO/AsGM1 mice are resistant to this mechanism of injury. To test these hypotheses, wild type and CD8KO/AsGM1 mice were treated with the broad-spectrum antibiotic imipenem immediately following CLP to decrease bacterial dissemination. Additional mice underwent cecal ligation without puncture of the cecal wall or cecal ligation followed by removal of cecal contents by irrigation of the cecal lumen. Treatment of wild type and CD8KO/AsGM1 mice with imipenem decreased bacterial counts by at least 2 orders of magnitude. However, all wild type mice, whether treated with saline or imipenem, died by 42 hours after CLP and had significant hypothermia, metabolic acidosis and high plasma concentrations of pro-inflammatory cytokines. Wild type mice subjected to cecal ligation without puncture also showed 100% mortality despite very low bacterial counts in blood. However, all wild type mice subjected to cecal ligation and washout of cecal contents survived. CD8KO/AsGM1 mice showed 50% long-term survival following CLP, which was increased to 100% by imipenem treatment. Following cecal ligation without puncture, CD8KO/AsGM1 mice showed 80 or 90% survival when treated with saline or imipenem, respectively. CD8KO/AsGM1 mice had less hypothermia, decreased metabolic acidosis and lower cytokine concentrations in plasma at 18 hours than wild type mice after CLP or cecal ligation without puncture. These results indicate that bacterial dissemination is not the major cause of mortality for wild type mice during rapidly lethal CLP. However, the presence of cecal flora is required for CLP-induced injury. CD8KO/AsGM1 mice appear resistant to early, non-infection-related, causes of CLP-induced injury but showed delayed mortality associated with bacterial dissemination, which was ablated by treatment with imipenem.