Transplantation antigenic activity was detected in the medium in which rabbit spleen cells had been cultivated in tissue culture. The antigenic material passed easily through a Millipore filter and could be recovered by ultracentrifugation. When injected i.d. into a recipient rabbit the antigenic material was consistently able to induce transplantation immunity as indicated by the accelerated rejection of a test skin homograft from the donor of the cultured spleen. That the antigenic activity was specific for the spleen donor could be demonstrated by histologic comparison of test skin homografts from the spleen donor and from an indifferent donor applied to the same antigen recipient. The antigenic preparation had no effect when injected back into the spleen donor. In its crude form the antigenic material was obtained as a fine suspension which was non-toxic upon i.v. infusion. Antigenic activity was stable to heating to 56 C, to freezing and thawing three times, to lyophilization, to storage at 4 C for 7 days, to pH 4.5, and to the action of trypsin. Detectable antigenicity was destroyed by heating to 90 C, by storage at 37 C for 4 days, by pH 12, and by exposure to periodate ion. The antigen appeared to be significantly more stable than several transplantation antigens recently extracted from mouse tissues by other workers.