At inflammatory sites, leukocytes may confront multiple, competing chemoattractive signals. We compared the chemotactic potencies of several sensory neuropeptides with regard to signal transduction pathways in eosinophils. Eosinophils were enriched using magnetic cell sorting and migration was assayed in a Boyden microchemotaxis chamber. We found stimulatory effects of substance P, calcitonin gene-related peptide (CGRP), secretoneurin, vasoactive intestinal peptide (VIP), and secretin on eosinophil migration. Actions of VIP are predominantly mediated via VIP receptor type I. Migration toward secretoneurin, VIP, and secretin was blocked by a phosphodiesterase inhibitor, which, in contrast failed to affect substance P- and CGRP-induced eosinophil chemotaxis. Wortmannin blunted the migratory responses induced by all neuropeptides tested and substance P-induced effects on eosinophils were tyrphostin-23-sensitive. We conclude that substance P, CGRP, secretoneurin, and VIP/secretin stimulate eosinophil migration involving wortmannin-sensitive enzymes. Moreover, secretoneurin and VIP/secretin require additional activation of phosphodiesterases to stimulate eosinophil migration.