Chemokine and integrin receptors must work in concert when circulating leukocytes mobilize toward a site of tissue inflammation or infection. In a previous study, we reported that ligation of the α5β1 integrin with a 120-kDa cell-binding fibronectin fragment (120-kDa FN) in suspensions of human polymorphonuclear leukocytes (PMNLs) inhibited chemotaxis toward the chemokine called interleukin-8 (IL-8). Binding of chemokines to their receptors on leukocytes leads to the activation of heterotrimeric G proteins that initiate multiple signaling cascades, including p38 and p42/p44 mitogen-activated protein kinase (MAPK) pathways. In the present study, we examine the potential interaction of β1 integrin ligation on chemokine-mediated MAPK signaling in human PMNLs. We demonstrate that blockade of the p42/p44 MAPK signaling pathway by the inhibitor PD98059 suppresses IL-8–mediated PMNL chemotaxis. Furthermore, when PMNLs are pretreated with 120-kDa FN or an activating antibody to β1 integrins (TS2/16), IL-8–mediated phosphorylation of p42/p44 MAPK is also inhibited. In contrast, pretreating PMNL with a specific ligand (laminin-1) for the α6β1 integrin does not suppress IL-8–mediated phosphorylation of p42/p44 MAPK. These observations demonstrate a desensitization of IL-8–mediated p42/p44 MAPK signaling in response to ligation of the α5β1 integrin in PMNL. Also, they suggest an interplay between integrin and chemokine signaling during PMNL migration through the extracellular matrix.