We have purified and characterized the guinea pig eosinophil chemotactic factor of anaphylaxis (ECF-A), an activity previously described in diffusates from sensitized lung challenged with specific Ag that appeared to selectively attract eosinophils from mixed leukocyte populations. Time course studies showed that the release of ECF-A from challenged presensitized guinea pig lung fragments closely paralleled the release of immunoreactive leukotriene B4 (iLTB4) and histamine. However, the majority of ECF-A (greater than 80%) and iLTB4 (greater than 79%) was extractable with the lipid fraction from the methanol wash of Sep-Pak-extracted diffusate, whereas histamine remained in the aqueous phase. A comparable neutrophil chemotactic activity was also found in the methanol extracts of the anaphylactic diffusates. By using a combination of HPLC and specific RIA, greater than 60% of ECF-A was attributable to LTB4. A second eosinophil chemotactic activity was also identified and coeluted (on both reverse phase and straight phase HPLC) with the synthetic standard 8(S),15(S)-dihydroxy-5,9,11,13(Z,E,Z,E)eicosatetraenoic acid (8(S),15(S)-diHETE). This was confirmed as 8(S),15(S)-diHETE by gas chromatography-mass spectrometry. Platelet-activating factor and histamine had negligible activity for guinea pig eosinophils, compared with synthetic LTB4 (p less than 0.05, 10(-9) and 10(-8) M; p less than 0.01, 10(-7) to 5 x 10(-6) M). In addition, synthetic 8(S),15(S)-diHETE had 3 times less activity than LTB4 at optimal chemotactic concentrations (10(-6) and 10(-7) M, respectively). Thus, guinea pig ECF-A appears to be largely attributable to lipoxygenase products of arachidonic acid, namely LTB4 and 8(S),15(S)-diHETE. Because guinea pig ECF-A was equally active on neutrophils (greater than 96% purity), it can no longer be considered a selective eosinophil chemoattractant.