Antigen challenge of the rat peritoneal cavity after passive preparation with IgGa-rich hyperimmune antiserum releases lipids that enhance the migration of human polymorphonuclear (PMN) leukocytes in a modified Boyden micropore filter assay. Deproteinated peritoneal fluid was applied to an Amberlite XAD-8 column equilibrated in distilled water and sequential elution yielded 30% of the migration-enhancing activity in water and 70% of the activity in 80% ethanol. The lipids in the 80% ethanol eluate were chromatographed on silicic acid that was developed sequentially with hexane, dichloromethane, acetone, 1-propanol, and ethanol:ammonia:water (6:3:1, v:v:v). The lipids with migration-enhancing activity eluted in hexane and acetone in two discrete peaks. These were separated from the other lipid mediators slow-reacting substance of anaphylaxis (SRS-A) and platelet-activating factor (PAF), which eluted in ethanol:ammonia:water. The activity in the acetone fraction had the functional characteristics of a chemotactic factor in that it required a concentration gradient to stimulate PMN leukocyte migration and induced chemotactic deactivation, whereas the hexane fraction contained a chemokinetic factor that enhanced PMN leukocyte migration irrespective of a concentration gradient. Both lipid factors were preferentially active on neutrophil and eosinophil PMN leukocytes as compared to mononuclear leukocytes. Reversed immunologic challenge of the rat peritoneal cavity with rabbit anti-rat F(ab′)2 antiserum released both the chemotactic and chemokinetic factors, whereas challenge with rabbit anti-rat myeloma IgE antiserum released predominantly the chemotactic factor. Pretreatment of the rat peritoneal cavity with 5,8,11,14-eicosatetraynoic acid (TYA) before direct immunologic challenge inhibited the release of both lipid factors and indomethacin pretreatment suppressed the immunologic release of the chemotactic factor, but not the chemokinetic factor. Thus, immunologic challenge of the rat peritoneal cavity by pathways dependent on antibodies other than IgE generates both PMN leukocyte chemotactic and chemokinetic activities from arachidonate or related fatty acids, whereas IgE-dependent stimulation releases predominantly the lipid chemotactic factor.