At inflammatory sites in vivo, leukocytes may confront multiple, competing chemoattractive signals. We found significant differences between eosinophils and neutrophils in transendothelial chemotaxis to a chemoattractant diffusing from the lower chamber, when a chemoattractant that binds to another receptor is present at uniform concentration. The transendothelial migration of eosinophils to FMLP, C5a, RANTES, or MCP-3 was totally inhibited by the presence of the homologous chemoattractant, and only RANTES and MCP-3 showed mutual inhibition. C5a and to a lesser extent FMLP chemokinetically stimulated migration to RANTES and MCP-3, without stimulating random migration. Results with neutrophils contrasted. The presence of FMLP not only abrogated neutrophil transmigration to FMLP but also strongly decreased chemotaxis to C5a, IL-8, and Gro-alpha. Similarly, C5a inhibited neutrophil chemotaxis to IL-8 and Gro-alpha. IL-8 almost totally abrogated chemotaxis to Gro-alpha, but Gro-alpha only moderately inhibited chemotaxis to IL-8. Neither IL-8 nor Gro-alpha significantly inhibited transmigration to FMLP or C5a. Actin polymerization in eosinophils and neutrophils was desensitized by the same combinations of chemoattractants that desensitized chemotaxis. We conclude that eosinophils have at least three noninterfering receptor-signal transduction pathways for chemotaxis and actin polymerization. In contrast, the signaling pathways for FMLP, C5a, and IL-8/Gro-alpha in neutrophils are heterologously cross-desensitized, with a hierarchy of resistance to competing signals of FMLP > C5a > IL-8 > Gro-alpha, in agreement with previous results in neutrophils on the Ca2+-mobilizing response. These results may have important implications for the behavior of these cell types in inflammatory sites.