Tumor cells sensitive to lysis by murine natural killer (NK) or natural cytotoxic (NC) cells were shown to bind laminin. They bound 125I-labeled laminin in a time- and concentration-dependent manner, and binding of the radioactive laminin was inhibited by excess cold laminin. In the presence of laminin, cell-cell aggregation occurred. Murine tumor cells not sensitive to NK/NC-mediated killing bound much less laminin, and laminin did not induce aggregation of these cells. The addition of exogenous laminin to NK or NC cytotoxicity assays reduced target lysis in a dose-related manner. Reduction of lysis was due to an inability of NK/NC cells to bind to the targets. Target cells pretreated with laminin were reduced in their ability to cold-target compete for NK-mediated lysis of untreated target cells. These effects were unique to laminin. The control proteins (fibronectin and thyroglobulin) had no effect on NK activity. Finally, inhibition of cytolytic activity by laminin appeared to be specific for NK/NC cells. Laminin had no effect on cytolysis mediated by alloimmune cytotoxic T lymphocytes regardless of whether the targets did or did not bind laminin.