In an attempt to study the effects of allogeneic lymphocytes on endothelial cells (EC) and analyze the mechanism whereby such lymphocytes traverse an EC barrier, we have established human microvascular EC monolayers, in vitro, and analyzed the effects of lymphocyte subpopulations on such monolayers. Previous studies have shown that CD16+ (natural killer) and CD8+ (cytotoxic) lymphocytes but not CD4+ (helper) cells bind and induce the appearance of class II major histocompatibility complex antigens on allogeneic EC. The current findings indicate that these same lymphocyte subsets induce marked swirling and elongation of allogeneic EC, and traverse intact EC monolayers. In contrast, none of the functional consequences of the initial lymphocyte-EC adhesion were observed using autologous combinations, despite the presence of significant intercellular binding. Scanning and electron micrographs demonstrate extensive areas of lymphocyte-EC surface contact and EC-coated pit formation, whereas a panel of recombinant cytokines known to alter the surface phenotype of EC fail to induce the same morphologic changes whether used singly or in combination. We postulate that the cellular interactions observed here, in vitro, may represent the initial steps in the rejection of vascularized allografts in vivo.