While a number of clinical studies indicate that elevated serum cytokine [interleukin-1 (IL-1), interleukin-6 (IL-6), and tumor necrosis factor (TNF)] levels are associated with enhanced mortality in sepsis, the time course and the role that different macrophage (Mφ) populations play in releasing these cytokines remain to be determined. To study this, polymicrobial sepsis was induced in C3H/HeN mice by cecal ligation and puncture (CLP). The animals were then sacrificed at 1, 4, or 24 hr post-CLP. Blood was taken for serum cytokine level determination. Macrophages, of either peritoneal (PMφ) or alveolar (AMφ) origin, were harvested by lavage, and their innate vs. inducible cytokine productive capacities were assessed by incubation with or without endotoxin (lipopolysaccharide; LPS). Serum levels of TNF were significantly enhanced 1 hr post-CLP (CLP = 3.8 ± 2.4* vs. sham = 0.4 ± 0.9 U/ml; *P < 0.05 by t test). However, not until 4 hr post-CLP were marked increases in IL-6 observed (CLP = 318.0 ± 209.0* vs. sham = 1.1 ± 0.5 U/ml), which remained elevated through 24 hr post-CLP (CLP = 11.3 ± 15.0* vs. sham = 0.03 ± 0.02 U/ml). Cytokine release (IL-1, IL-6, TNF) from PMφ (without the addition of LPS) was detectable only in cells harvested 1 h following CLP. Alveolar Mφ from septic mice showed little in vivo activation. Septic PMφ IL-1 and IL-6 production was markedly depressed at all time points with LPS stimulation, but TNF release remained unaltered. Conversely, AMφ showed a significant decline in TNF production at 1 hr post- CLP, while IL-1 and IL-6 levels were depressed at 24 hr. Thus the marked early (1-4 hr post CLP) elevation in the cytokine levels in the blood appear to be associated with in vivo activation of PMφ but not AMφ. However, the blunting of LPS-inducible PMφ cytokine production may be related to developing LPS tolerance or may be the result of direct cellular injury associated with the developing septic state.