Paraformaldehyde-fixed mKSA tumor cells of BALB/c mice were shown to retain tumor-associated transplantation antigen (TATA) activity to a degree comparable to that of X-ray-inactivated tumor cells under the optimal conditions of 1% paraformaldehyde for 30 min at 37 degrees. Unexpectedly, the TATA activity of cells fixed below 10 degrees was greatly reduced. This temperature effect was reversible. TATA activity was restored if the cells were returned to 37 degrees before fixation. Fixation at all temperatures for longer than 2 hr or at paraformaldehyde concentrations greater than 1% also caused a decrease in immunogenicity. Spleen cells from mice immunized with tumor cells fixed at 37 degrees were able to more effectively neutralize tumor growth in the Winn assay compared with those from mice immunized with cells fixed at 0 degrees. Immunization with paraformaldehyde-fixed tumor cells was completely specific. Mice immunized with an antigenically unrelated tumor were not rendered immune to tumor challenge. Fixed tumor cells could be stored for at least 1 month without loss of TATA activity.