The sequence of rat alpha-calcitonin gene-related peptide (CGRP-alpha) contains the tetrapeptide eosinophil granulocyte chemotactic factor Val32-Gly-Ser-Glu35. Peptide fragments formed following hydrolysis of rat CGRP-alpha in vitro by endopeptidase-24.11 were identified. The tetrapeptide fragment was generated following cleavage at a substrate recognition site unusual for this enzyme (-Glu-Ala-). Chemotactic activity of rat CGRP-alpha was increased following hydrolysis. Furthermore, rat CGRP-beta, which lacks the tetrapeptide sequence and is completely devoid of chemotactic activity, displayed low but measurable activity after hydrolysis. Val-Gly-Ser-Glu was identified as the principle fragment with chemotactic activity in rat CGRP-alpha. The results show that the chemotactic activity of the neuropeptide rat CGRP-alpha towards eosinophil polymorphonuclear leukocytes is increased following its hydrolysis in vitro by endopeptidase 24.11 through the formation of a previously identified eosinophil chemotactic tetrapeptide.