The chief impediment to the precise biochemical identification of transfer factor and its mechanism of action has been the lack of a reproducible in vitro assay. We now report on a method by which dialysates containing transfer factor of proven in vivo potency can convert nonimmune lymphocytes to immune responsiveness in vitro, as reflected by antigen-triggered lymphocyte proliferation. However, "water-dialyzed transfer factor" (TF(D)) prepared for in vivo use by the conventional method of dialysis against large volumes of water exhibits diminished activity in vitro and is frequently toxic to lymphocyte cultures. This problem can be avoided by dialysis of transfer factor into tissue culture medium. When this precaution is taken, such "media-dialyzed transfer factor" (TF(DM)) causes nonimmune lymphocytes to respond to antigen by an increment of thymidine incorporation that ranges from 2 to 25 times that of such cells cultured with antigen alone. This response is generally observed only in the presence of those antigens to which the TF(DM) donor expresses delayed cutaneous reactivity and is distinguishable from nonspecific adjuvant effects.