The finding of outer membrane protein I (OprI) of Pseudomonas aeruginosa in hemodialyzers used by patients with end-stage renal failure led us to study the possible role of OprI as cytokine inducer. However, there are few reports on the biological activity of OprI, because it is difficult to obtain highly purified OprI. In this study, we attempt to establish a procedure for the efficient purification of OprI, which does not include lipopolysaccharide, from the bacterial culture broth, not hemodialyzers, to demonstrate that OprI is a potent cytokine inducer. From bacterial culture broth (1 liter), P. aeruginosa PAO1, which was confirmed previously by the sequence coding, was separated by centrifugation, high-performance liquid chromatography, and disk electrophoresis. Mouse bone marrow cells were stimulated by purified OprI, and the supernatants of the culture were analyzed by several enzyme-linked immunosorbent assay kits. The tumor necrosis factor alpha production stimulated by purified OprI was confirmed and degraded within 24 h. Furthermore, interleukin (IL) 1α, IL-1β, IL-6, and granulocyte-macrophage colony-stimulating factor were also induced by OprI despite the absence of lipopolysaccharide. We conclude that OprI has the potential to induce tumor necrosis factor alpha production in mouse bone marrow cells and that tumor necrosis factor alpha contributes to the induction of inflammatory cytokines, namely IL-1α, IL-1β, IL-6, and granulocyte/macrophage colony-stimulating factor, while lipopolysaccharide has little effect on these cells. These results suggest the presence of a pathway of inflammatory signal transduction triggered by OprI. In addition, OprI is possibly one of the harmful dialysate pollutants in hemodialysis patients besides the well-known lipopolysaccharide.