Polymyxin nonapeptides, proteolytic derivatives of polymyxin antibiotics, are less toxic than their parent compounds but retain some of their antibacterial activities. To confirm and expand observations that polymyxin nonapeptides have anti-endotoxin activity, we studied the ability of colistin nonapeptide to bind to bacterial lipopolysaccharide (LPS) and to inhibit the effects of LPS on Limulus amoebocyte lysate and lymphocyte mitogenicity. Colistin nonapeptide was purified by high-pressure liquid chromatography and was demonstrated to bind to LPS by equilibrium dialysis. The ability of colistin nonapeptide to render E. coli ATCC 25922 cells sensitive to erythromycin was abrogated by 50% after incubation with E. coli O18 LPS in a ratio by weight of LPS to colistin nonapeptide of 3.9:1. The presence of 4 micrograms of colistin nonapeptide or colistin per ml increased by 130- and 800-fold, respectively, the concentration of E. coli O113 LPS required to produce 50% gelation of Limulus amoebocyte lysate as measured by a spectrophotometric assay. Neutralization of LPS by colistin nonapeptide was time and concentration dependent. In contrast to the neutralization seen with LPS derived from a colistin-sensitive organism, colistin nonapeptide neutralized very little LPS extracted from a strain of Serratia marcescens that was resistant to colistin. Colistin nonapeptide also inhibited LPS-induced [3H]thymidine uptake by splenic lymphocytes, but its activity was less than 1/10 that of colistin. We conclude that colistin nonapeptide binds to LPS and possesses antiendotoxin activity. However, the anti-endotoxin activity of the nonapeptide is considerably less than that of its parent compound, colistin.