Guinea pig peritoneal eosinophils stimulated by platelet-activating factor (PAF), leukotriene B4 (LTB4), and human recombinant C5a (C5a) undergo a rapid concentration-dependent and partially reversible homotypic aggregation as assessed by changes in light transmission. The phorbol ester phorbol myristate acetate similarly induces a concentration-dependent aggregation, which is, however, slower in onset, takes longer to reach maximal aggregation, and is irreversible. In addition, we confirmed, using light microscopy, that these agonist-induced changes in light transmission do indeed represent true homotypic aggregation. We further characterized the aggregation response and showed that there is homologous but little heterologous desensitization when PAF and LTB4 are used as stimuli. A requirement for both Ca2+ and Mg2+ for full manifestation of agonist-induced aggregation was observed. LTB4- and PAF-induced superoxide anion generation is enhanced by the diacyglycerol kinase inhibitor R59022, whereas aggregation induced by LTB4, but not PAF, is augmented. Lastly, we show that eosinophil aggregation is partially dependent on the adhesion glycoprotein CD18. In summary, therefore, we believe that eosinophil aggregation provides a useful and reliable measure of eosinophil activation.