During extravasation and within lymph nodes (LNs), blood lymphocytes interact with laminins (Lms), major components of vascular basement membranes (BMs) and of reticular fibers (RFs), a fibrillar extracellular matrix. However, the identity and role of these laminin isoform(s) are poorly known. By using confocal microscopy examination of human LNs, we show that BMs of high endothelial venules (HEVs) express laminin α3, α4, α5, β1, β2, and γ1 chains and that the same chains, in addition to α2, are found in RFs. In functional studies with laminin isoforms covering all Lm α chains, α5-laminin (Lm-511) was the most adhesion- and migration-promoting isoform for human blood lymphocytes, followed by α3- (Lm-332) and α4- (Lm-411) laminins, and the lymphocytes used the α6β1 integrin as the primary receptor for the α5-laminin. Moreover, Lm-511 strongly costimulated T cell proliferation, and blood lymphocytes were able to secrete α4- and α5-laminins following stimulation. The LN cell number in laminin α4-deficient mice compared with wild-type did not differ significantly. This study demonstrates a predominant role for α5-laminin(s) in blood lymphocyte biology and identifies LN laminins and their integrin receptors in blood lymphocytes.