Peptidoglycan (PG) is the major component of the cell wall of gram-positive bacteria. In vitro, PG isolated from conventional bacterial cultures can induce secretion of proinflammatory cytokines by human monocytes, indicating that PG may be involved in immune responses against infections by gram-positive bacteria. To investigate the biologic activity of PG in human tissues, an improved method was developed to isolate significant amounts of PG from sterile human spleen tissue. Biochemical analysis demonstrated that PG isolated from human spleen is largely intact. Human whole blood cell cultures were able to produce the proinflammatory cytokines tumor necrosis factor-alpha and interleukin-1 and -6 after stimulation with PG isolated from human spleen. Cytokine induction was not sensitive to inhibition by polymyxin B, in contrast to lipopolysaccharide. Collectively, the data show that intact PG in sterile human tissue is biologically active and may induce local proinflammatory cytokine production.