The presence of small amounts of endotoxins are often undesirable when investigating cytokines such as interleukin-1 and tumor necrosis factor alpha. Polymyxin B, widely used to block endotoxins, does not block several forms of endotoxins, and at high concentrations, polymyxin B itself stimulates interleukin-1 production. Human peripheral blood mononuclear cells are highly sensitive to endotoxins; they respond with cytokine production to endotoxins at concentrations of 10-50 pg/ml and detect pyrogenic materials nonreactive in the Limulus test. In the present study, ultrafiltration using polysulfone filters was found to remove all interleukin-1- and tumor necrosis factor alpha-inducing substances produced in E. coli cultures. Interleukin-1- and tumor necrosis factor alpha-inducing substances derived from Pseudomonas aeruginosa cultures were also rejected by the filters. Ultrafiltration is therefore a convenient and effective procedure to remove interleukin-1 and tumor necrosis factor alpha-inducing substances from parenteral fluids and solutions that come in contact with blood such as fluids used in hemodialysis. This technique is also applicable for the large-scale production of culture media for mammalian cell expression of recombinant, pyrogen-free proteins intended for use in humans.