Exocytosis of granules containing the cytolytic effector (CE) molecules granzyme A (GzmA), granzyme B (GzmB), and perforin is one major pathway of lymphocyte-mediated cytotoxicity. Studies in murine models and the finding of elevated granzyme levels in the plasma of septic patients have implicated cytotoxic lymphocytes in the pathogenesis of sepsis. We sought to evaluate the role of cytotoxic cells and CE in sepsis and determine if intracellular levels of CE in cytotoxic cells correlate with disease severity. We conducted a prospective cohort study of 40 patients enrolled into one of three groups: controls (C), acutely ill nonseptic illnesses, or patients with severe sepsis (SS) (lactate, >4 mmol/L; systolic blood pressure, <90 mmHg after 2 L normal saline). Peripheral blood mononuclear cells were isolated and stained for extracellular markers for defined subpopulations and for intracellular expression of GzmA and GzmB and perforin. Levels of CE were quantified by geometric mean fluorescent intensity (GMFI) via flow cytometry. Cytotoxic T lymphocyte (CTL) expression was higher in SS (P = 0.04). The GMFI of GzmB was significantly higher in CTLs of SS patients versus acutely ill nonseptic illnesses or C. The GMFI of each GzmA and GzmB in CTLs were associated with the Acute Physiology and Chronic Health Evaluation II score (P = 0.01). A significant increase in the number of granulocytes in the peripheral blood mononuclear cells of SS patients consisted primarily of low-density neutrophils, which expressed increased levels of GzmA (P < 0.01). The results suggest that CTLs are activated in SS and express significantly higher intracellular levels of GzmB and that GzmA and B levels correlate with disease severity.