We previously reported that the prophylactic, intraperitoneal administration of supernatants from concanavalin A-stimulated T cells derived from Salmonella enteritidis (SE)-immune White Leghorn hens (i.e., SE-immune lymphokines or ILK), conferred protection to neonatal White Leghorn chicks against SE organ invasion. In the present study, we evaluated the effects of in ovo administration of ILK on hatchability, hatch weight, in vitro bactericidal activity of heterophils, and protection against SE organ invasion in neonatal White Leghorn chicks. On Day 18 of embryogenesis, injections were made into the amnion with either ILK or nonimmune ILK (NILK) or were not injected (untreated). On the day of hatch, whole blood was collected from 20 of the chicks per treatment group for heterophil isolation. All remaining chicks were orally challenged with 5 x 10(4) cfu SE. Twenty-four hours after SE challenge, organs (liver and spleen) from the chicks were cultured for SE. Hatchability of ILK- and NILK-treated chicks was not different from that of untreated chicks. Hatch weights of ILK-treated chicks were approximately 1 g less (P < .05) than that of NILK-treated or untreated chicks. In vitro bactericidal activity of peripheral blood heterophils derived from ILK-treated chicks was increased (P < .05) above activity from heterophils derived from NILK-treated or untreated chicks. Organ invasion with SE was markedly and significantly decreased in the ILK-treated chicks as compared with chicks treated with NILK or untreated chicks. These results suggest that in ovo administration of ILK confers protection to neonatal chicks against SE organ infectivity at hatch.