We have studied the mechanism of the cell-mediated mitogenic response (CMMR), in which proliferative responses are generated in mouse T lymphocytes co-cultivated with syngeneic mitomycin C-treated spleen cells (Mito-SP), when either responder or stimulator cells are briefly pretreated with concanavalin A (Con A) under nonmitogenic conditions. We present evidence that an intact membrane of the stimulator cells is required in CMMR, since the response was abolished by fixation, or by freezing and thawing of stimulator cells. The fate of cell-bound Con A was studied by tracing I-Con A bound to either stimulator or responder. A lymphoblast that developed from untreated CRT stimulated by I-Con A-coated syngeneic Mito-Sp carried about 105 molecules of 125I-Con A. The amount of Con A released into the culture medium was not sufficient for inducing a mitogenic response by itself, nor to bind to cells at the level found in CMMR, suggesting that 125I-Con A was transferred directly from labeled cells to unlabeled cells. Transferred 125I-Con A found in lymphoblasts was undergraded intact Con A, as demonstrated by gel electrophoresis. Autoradiography allowed visualization of the movement of 125I-Con A from stimulator to responder cells within 60 min of cell contact.