Sensitization of lymphocytes against allogeneic cells in vitro results in significant enrichment of alloreactive lymphocytes. Such enrichment was found to profoundly influence the conditions for measuring proliferative responses. Fewer primed lymphocytes than unprimed ones had to be cultured to favor optimal proliferation. Second, proliferative responses could be detected using 10 to 20 times fewer primed responding cells than when using unprimed responders. Finally, although responses of both unprimed and primed lymphocytes were dependent on the number of stimulator cells in culture, the primary mechanism(s) through which this dependence was expressed appeared to differ. The results demonstrate that, under the same conditions, comparisons of responses of two populations that contain different proportions of reactive lymphocytes may not be justified.