We have adapted a chemotaxis assay using human umbilical vein endothelial cell (HUVEC) monolayers on microporous membranes for studying lymphocyte transendothelial chemotaxis in vitro. Supernatants of peripheral blood mononuclear cells stimulated with phytohemagglutinin (PHA) were identified as an excellent source of lymphocyte chemoattractant activity. The activity in PHA supernatant typically caused 2-6% of peripheral blood lymphocytes (PBL) to transmigrate compared to 0.1-0.3% to media control. Checkerboard analysis demonstrated that transmigration was directional and not attributable to random locomotion. Purified T lymphocytes also underwent transendothelial chemotaxis to PHA supernatant. Using monoclonal antibodies to several human adhesion receptors, we found that the interaction between LFA-1 and ICAM-1/ICAM-2 was more important for transendothelial lymphocyte chemotaxis than the interaction between VLA-4 and VCAM-1. A monoclonal antibody to the beta 1 integrin subunit inhibited chemotaxis more than antibodies to the VLA alpha 2, alpha 3, alpha 4, or alpha 5 subunits. The transendothelial assay was used to guide purification of the lymphocyte chemoattractant activity, which we reported previously to be monocyte chemoattractant protein-1 (MCP-1) (Carr et al., Proc. Natl. Acad. Sci. USA (1994) 91, 3652). The adhesion molecules required for chemotaxis to MCP-1 were similar to those with PHA supernatant. The use of HUVEC in the assay enhances the signal-to-background ratio of chemotaxis and provides a model that is physiologically relevant to lymphocyte emigration from the bloodstream into sites of inflammation.