The effect of Pseudomonas aeruginosa heat-stable hemolysin (rhamnolipid) on human neutrophil migration has been investigated. Rhamnolipid was prepared from culture filtrate and characterized by thin-layer chromatography. The lytic activity of rhamnolipid was quantitated by titration against neutrophils. Leukocyte migration response was measured using51Cr-labeled neutrophils with a double-filter technique in modified Boyden chambers. The results suggest rhamnolipid stimulated chemotaxis as well as chemokinesis. Moreover, rhamnolipid impaired a chemotactic response to N-formyl-methionyl-leucyl-phenylalanine. These effects may be important in host-parasite interactions.