In combination with xenogeneic immune RNA (l-RNA) and tumor antigen (TA), syngeneic spleen cells inhibited the growth of a N-methyl-N-nitrosourethane-induced colon carcinoma (CT-26) in BALB/c mice. The sequential administration of 1 X 10(7) spleen cells, l mg of anti-CT-26 l-RNA and 0.4 mg of CT-26 TA resulted in a 20% survival rate of mice bearing established CT-26 tumors. On the other hand, administration of the spleen cells only, l-RNA only or TA only was not effective in inhibiting tumor growth. Similarly, when any one of the three agents (l-RNA, TA and spleen cells) was omitted, no anti-tumor effect was obtained. A higher dose (1 X 10(8) and a lower dose(1 X 10(6) of spleen cells decreased the anti-tumor effect of this combination therapy. A higher dose (2 mg) and a lower dose (0.5 mg) of l-RNA, as well as a higher dose of TA (0.8 mg) had no influence on the anti-tumor effect. However, a lower dose of TA (0.2 mg) in combination with spleen cells and l-RNA decreased the anti-tumor effect. When all three agents were administered at higher or lower doses, no anti-tumor effect was obtained. When mice bearing CT-26 tumors were treated with spleen cells, plus l-RNA directed against a syngeneic but antigenically different tumor (BP/B/5) and BP/B/5/TA, with spleen cells plus CT 26 I-RNA and BP/B/5TA, or with spleen cells plus BP/B/5 l-RNA and CT-26 TA, no anti-tumor effect was observed. These results indicate that the anti-tumor responses observed were due to tumor-specific immune responses. In conclusion, in order to obtain growth retardation or regression of established CT-26 tumor transplants, the sequential administration of all three agents (spleen cells, l-RNA and TA) was required. The optimal doses of each agent were found to be 1 X 10(7) spleen cells, 0.5 mg or 1 mg of l-RNA and 0.4 mg of tumor antigen. Higher or lower doses decreased the anti-tumor effect. Tumor-specific immune reactions appeared to be involved.