The effect of polymyxin B (PMB) on the endogenous pyrogen (EP)-induced property of lipopolysaccharide (LPS) in vitro was examined. PMB inhibited LPS when added to leukocyte suspension 5 min before or up to 30 min after the addition of LPS. The inhibitory effect was dose-related and appeared to be specific for LPS (including naturally occurring endotoxin). EP production in response to a different stimulus (staphylococci) was not prevented even when LPS-PMB complexes were presumably present. These data suggest that when experimental agents are found to stimulate the production of EP or lymphocyte activating factors (LAF, interleukin-1) in vitro, or when apparently spontaneous production of EP or LAF is seen, incubation with PMB may be a useful technique to exclude th effects of endotoxin contamination - especially when negative results have been obtained in the limulus gelation test.