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Notes: Summary The therapeutic effect of i. d. injection of tumor cells mixed with Vibrio cholerae neuraminidase (VCN) on tumor progression in dogs with spontaneous mammary tumors was investigated. The i. d. injections were performed in a chessboard-like manner: different numbers (105, 106, 107, and 108) of mitomycin-treated autologous tumor cells (M-TC) were each mixed with different amounts (10, 50, and 100 mU) of VCN. These different mixtures were injected i. d. at different sites in dogs on the day of resection of a part of multiple tumors. In a randomized prospective study in 71 dogs the effect of chessboard vaccination (autologous tumor cells and VCN) on the growth of the residual tumor mass was compared to chessboard-like treatments with mixtures of either autologous erythrocytes and VCN or autologous tumor cells and heat-inactivated VCN. The results show that: chessboard vaccination induced regression (6 of 23) of spontaneous mammary tumors in dogs. No dog died as a result of the tumor within an observation period of 1 year. The therapeutic effect of chessboard vaccination was dependent on the application of tumor cells and enzymatically active VCN. In contrast, control treatment with either heat-inactivated VCN or autologous erythrocytes instead of tumor cells did not induce any regressions. Some animals in both control groups died because of tumor growth (3/21 and 2/27 respectively). The delayed type hypersensitivity (DTH) response of tumor-bearing animals against i. d. applied tumor cells was not significantly enhanced by the admixture of enzymatically active VCN, nor did the DTH response seem to be predictive of a therapeutic effect on the tumor. No difference in the DTH response of dogs to autologous tumor cells mixed with active or inactivated VCN or autologous erythrocytes mixed with active VCN could be found. Thomsen-Friedenreich antigens were serologically detected on canine erythrocytes after treatment with VCN and on untreated cells of mammary tumors from dogs. Exposure of Thomsen-Friedenreich antigens after treatment with VCN was enhanced on canine mammary tumors. As chessboard vaccination proved to be unsuccessful when canine autologous erythrocytes were used instead of autologous tumor cells, it can be concluded that the exposure of Thomsen-Friedenreich antigen plays no decisive role in tumor therapy with tumor cells and VCN. Chessboard vaccination was tolerated without any side effects. Tumor enhancement was never observed. Chessboard vaccination appears to be an effective and safe procedure for tumor therapy using tumor cells and VCN. The mechanism underlying the therapeutic effect of chessboard vaccination is completely unknown.