Potentiation by cytochalasin B (CB) of (3H)thymidine incorporation in lymphoid cell suspensions stimulated by phytohemagglutinin (PHA) is counteracted by adherent cells. The drug has no effect on the production of the macrophage product lymphocyte activating factor (LAF) and does not interfere with its potentiating activity. CB appears to inhibit agglutination of lymphocytes. Other factors (absence of serum, macrophages, mechanical disturbance of the cultures) diminishing the size of aggregates increase the response of lymphocytes. Responses to less agglutinating mitogens are less or not enhanced by CB. Factors increasing agglutination or/and cell contacts (high cell density, wheat germ agglutinin) diminish lymphocyte activation and make it susceptible to CB potentiating effect. The results suggest that topoinhibition (cell contact inhibition) is an important mechanism regulating the lymphocyte response to mitogens in culture. Its effect could be modulated by macrophages and certain serum factors, and CB would potentiate these responses by reducing contact inhibition.