The minimum length of time required for lymph node lymphocytes to recognize alloantigens in vitro was examined in mixed skin cell-lymphocyte culture reactions. Responding lymphocytes in the mixture were successfully separated from stimulating skin cells by Ficoll-Hypaque gradient sedimentation. Lymphocyte activation by allogeneic skin cells took place within 15 min of contact. The amount of measured stimulation was approximately 30% of that produced by an equal concentration of antigen present continuously in culture. The presence of alloantigen was no longer needed after 24 hr. Several control experiments were made in order to exclude the effects of contamination and culture conditions. Identical results were obtained with allogeneic hepatocytes. These data indicate that the recognition of alloantigen leading to lymphocyte proliferation may occur during the early period of contact.