Summary Following experiments which suggested that apparent stimulation of mouse spleen cells by fetal calf serum (FCS) was masking specific blastogenic effects in the mixed leukocyte culture (MLC), cultures of CBA and BALB/c spleen cells were set up in RPMI-1640 medium, containing FCS at 5, 4, 3, 2, 1 and 0%. On termininating the cultures on day 4, the ratio of 3H-thymidine incorporation between the mixed and the separate cultures was found to be considerably higher in absence of FCS than in the presence of FCS at any of the concentrations indicated. A series of timed cultures, without any FCS, showed a ratio between the incorporation of 3H-thymidine in the mixed cultures and the sum of incorporation in cultures of separate cells which reached a maximum level of 11-15, on day 5 or 6. In tests with cells of other sources, it was found that thymus cells could serve as a source of allogeneic antigen, but could not incorporate thymidine, and that bone marrow cells could be stimulated, but to a lower degree than spleen cells,