Chemokine receptors and adhesion molecules are used selectively for the transmigration of leukocytes across the blood-brain barrier (BBB) during neuroinflammation. We established an activated in vitro BBB (aIVBBB) using physiological concentrations of cytokines. We studied CCL5-driven migration as a model to determine how chemokine receptors and adhesion molecules regulate T-cell and monocyte migration across the aIVBBB. Increased expression of CCL5 and its receptors, CCR1 and CCR5 have been described in the perivascular space of multiple sclerosis (MS) lesions. Elucidating the determinants of CCL5-mediated mononuclear cell migration may clarify appropriate targets for therapeutic modulation in neuroinflammatory conditions. In response to CCL5, there was a significant increase in total mononuclear cell migration across the aIVBBB. Neutralizing monoclonal antibodies to CCR1 and CCR5 abrogated CCL5-driven transmigration, suggestive of non-redundant receptor usage in mononuclear cell migration to this chemokine in vitro. CCL5-driven transmigration was also dependent on alpha(4)beta(1) integrin/fibronectin connecting segment-1 (FN CS-1) and alpha(L)beta(2) integrin/intercellular adhesion molecule (ICAM-1) interactions. Monocyte migration to CCL5 was solely dependent on alpha(4)beta(1) integrin/FN CS-1 while T-cell migration required both alpha(L)beta(2) integrin/ICAM-1 and alpha(4)beta(1) integrin/FN CS-1 interactions. These findings provide plausible molecular targets for the selective inhibition of mononuclear cell trafficking during the acute immune effector phases of MS and other neuroinflammatory diseases.