We investigated the changes in neutrophil phenotype and function after transendothelial migration, and the roles played by integrin receptors in their behaviour. Neutrophils were tracked microscopically as they migrated through endothelial cells into collagen gels, and were retrieved at desired times. When endothelial cells were treated with increasing doses of tumour necrosis factor-α, neutrophils not only migrated in greater number, but also to a greater depth in the gel. Apoptosis was barely detectable in neutrophils retrieved after 24h, and many remained viable and motile at 48h. Neutrophils retrieved after 1h had increased oxidative capacity and at 24h had similar capacity as freshly-isolated neutrophils. However, by then they had impaired ability to phagocytose bacteria. Compared to fresh neutrophils, total mRNA was halved by 24h, but while β2-integrin expression decreased, β1- and β3-integrin increased along with ICAM-1. Studies of integrin blockade indicated that while β2-integrins were needed to cross the endothelial barrier, no integrins were required for migration within the gel. β2-integrins also contributed to phagocytosis, but their binding was not required for prolonged survival. These results demonstrate a model for integrated analysis of neutrophil migration and function, and describe development of effector functions and the roles of integrins in human neutrophils for the first time.