Antitumor immunity requires (a) extravasation of lymphocytes from the blood stream to interstitium, (b) locomotion through extracellular matrix to the site of the tumor, (c) effector cell recognition of the tumor target with cell/cell contact and binding of adhesion receptors, (d) T-cell receptor binding to histocompatibility and tumor antigens, and (e) tumor cell lysis. We hypothesize that the tumor microenvironment inhibits lymphocyte locomotion through extracellular matrix as one mechanism by which tumors may avert host defense. Lymphocyte locomotion was investigated in vitro using a three-dimensional collagen gel model. Fresh tumor-infiltrating lymphocytes (TIL) were obtained by enzymatic digestion of melanomas and renal cell carcinoma, and mononuclear cells were isolated by discontinuous Ficoll-Hypaque gradient. The lymphocytes were analyzed for motility from a point of origin between basal and overlay layers of collagen gel. Results showed that TIL migration was almost completely inhibited, compared with migration of normal and cancer patient peripheral blood leukocytes and lymphocytes from lymph nodes. Short-term (24-h) exposure of lymphocytes to cytokines during the assay in the collagen gel matrix had no effect on locomotor ability. Long-term (19, 30, or 35 days) culture of TIL in 200 units/ml of interleukin 2 reinstated locomotor ability. Short-term exposure of any of the lymphocyte populations to interleukin 1-alpha, interleukin 1-beta, interleukin 2, interleukin 3, interleukin 4, alpha-interferon, or gamma-interferon had no effect on migration. Thus, TIL display a uniquely arrested ability to locomote through collagen gel. Inhibition of the locomotion of infiltrating effector cells is possibly a mechanism by which the tumor evades the host immune system.