Our laboratory has previously isolated several tumor cell populations from a single, spontaneously arising mammary tumor of a BALB/cfC3H mouse and established them in tissue culture as independent sublines. These subpopulations differ according to many criteria including growth parameters and expression of tumor-associated antigens. We have tested the interaction in vivo of several of these subpopulations by injecting cell suspensions of the same or different sublines into opposite flanks of BALB/cfC3H or BALB/c mice. The growth characteristics of certain subpopulations were altered by the presence of a different subpopulation on the opposite side. In order to understand the mechanism of interaction, we chose two subpopulations (410 and 168) for further study. In BALB/cfC3H mice, the presence of line 410 tumors on one flank inhibited both 410 and 168 tumors on the other flank. Line 168 tumors did not inhibit either 410 or 168 tumors. The inhibitory effect of line 410 appeared to be immunological, since (a) it was increased by injecting line 410 several weeks before line 168, (b) it was abrogated in mice subjected to 400-rad X-irradiation 2 days prior to tumor cell injection, (c) mice could be made resistant to both line 410 and line 168 tumors by implantation followed by surgical removal of line 410 but not of line 168, and (d) resistance could be adaptively transferred with lymph node cells from line 410-sensitized mice in Winn assays. Thus, immunity to tumor-associated antigens may be one way by which cells of a heterogeneous tumor can interact.