The mitogenic response in vitro of DA rat splenic lymphocytes to concanavalin A has been found to be greatly enhanced (up to 800-fold) if the in vitro environment in which the cells are cultured is modified by the addition of nonmitogen-responsive, mitomycin C-treated “filler” cells. The results of the experiments suggest that filler cells may act as “spacer” cells and that the phenomenon is a physical effect in which the additional cells act as non-immunological cushions that modulate suppressive factors limiting cell responsiveness in vitro. Cell viability of the spacer cells was not necessary and the enhanced responses that follow the addition of spacer cells could be duplicated by formalin-fixed cells or even non-biologically active material such as Sephadex or Bio-Gel. Soluble factors released from spacer cell preparations also resulted in a modest increase in mitogenic responsiveness. The experiments further define the conditions for the culture of rodent lymphocytes and underscore the need for controls that eliminate nonbiological effects as explanatory mechanisms where cell collaboration is putatively involved in the generation of cell-mediated immune responses.