Spleen cells derived from tumor-bearing mice prove useful for the elucidation of the mechanism determining how tumor cells evade cytotoxic T lymphocytes (CTL) in tumor-bearing hosts. Our data indicate that inactive CTL or precursor CTL specific for tumor antigens are present among lymphocytes of tumor-bearing mice. However, their activity is inhibited by a soluble factor produced by other cells present in the same source. Inhibition of the cytolytic reaction was also detected in the culture supernatant of spleen cells obtained from normal mice, precultured in the presence of tumor cell culture supernatant and interleukin-2 (IL-2). Cell-depletion and cell-purification studies let us conclude that cells that produced the CTL-inhibitory factor (CTL-IF) were gamma/delta T cells. The gamma/delta T cells that were activated in vivo in tumor bearers were able to produce CTL-IF after isolation and in vitro culture. Maximum activation of gamma/delta T cells was achieved by antigenic stimulation and by suppression of cells that interfered with the activation of gamma/delta T cells. CTL-IF, which was assayed by use of CTL clones, did not show antigen specificity. Inhibition depended on a relatively heat- and acid-stable, but alkali-labile molecule with a molecular mass of less than 10 kDa. The latter characteristics imply that CTL-IF does not resemble any of the known lymphokines produced by gamma/delta T cells. These observations emphasize the crucial role of the gamma/delta T cells in the escape of tumor cells from the attack of tumor-specific CTL.