Hematopoietic colony stimulating factors (CSF) regulate the growth and development of phagocytic cell progenitors and also augment functional activation of phagocytes. Granulocyte-CSF (G-CSF) is the CSF that acts specifically upon granulocyte progenitor cells and mature granulocytes. We have shown that lymphokines (ILK) from T cells of birds immunized against Salmonella enteritidis (SE) induce a granulocytic (PMN) inflammatory response in chicks challenged with SE. This inflammatory response was characterized by: (a) a dramatic emigration of granulocytic cells from the bone marrow into the peripheral blood, (b) an enhancement of the biological functions of the circulating PMNs, and (c) a directed influx of these activated PMNs to the site of bacterial invasion. In the current study, we determined the presence of G-CSF in ILK by Western blot analysis using a goat polyclonal antihuman G-CSF antibody (Ab). Using this Ab, we then evaluated the role of G-CSF in the ILK-induced protective inflammatory response in chickens against SE. Pretreatment of ILK with the Ab totally abolished the colony-stimulating activity of the ILK. Furthermore, Ab treatment of ILK resulted in: (a) an elimination of the ILK-induced peripheral blood heterophilia with a dramatic inhibition of ILK-mediated protection against SE organ invasion and (b) an elimination of accumulation of inflammatory PMNs in the peritoneum with subsequent decrease in the survival rate of chicks challenged i.p. with SE. Taken together these studies demonstrate for the first time the contribution of G-CSF to avian PMN activation and the immunoprophylaxis of SE infection by ILK in neonatal chickens.