We examined the killing activity of transmigrated lymphokine-activated killer (LAK) cells and their surface molecules associated with both transendothelial migration and cytotoxicity, using human umbilical vein-derived endothelial cell (HUVEC) monolayers on fibronectin with gelatin separating the upper chamber from the lower chamber. Migratory LAK cells were significantly more cytotoxic to Daudi target cells, expressed more LFA-1, and were more likely to be positive for CD2, compared to those LAK cells not adherent to the HUVEC monolayer. In contrast, in the absence of the HUVEC monolayer, there was no difference in LAK activity between migratory and non-adherent LAK cells. These results indicate that the interaction between LAK cells and the HUVEC monolayer allows selective migration of LAK cells with cytotoxic activity that is enhanced with respect to some surface molecules.