The double layer soft agarose clonogenic assay using colony formation of target cells as an endpoint was adapted for the detection of antiproliferative lymphokine production from peripheral blood lymphocytes (PBL) and lymph node lymphocytes (LNL). The colony formation of cervical cancer cell lines, HeLa, CAC-1, and TMCC, in the upper layers was significantly inhibited by the inclusion of either PBL or LNL pretreated with PHA in the lower layers. Without stimulation by PHA, neither resident PBL nor LNL exhibited antiproliferative activity on the tumor cells in the upper layers. The antiproliferative activity against target cells increased in relation to the density of lymphocytes in the lower layers, and was dependent on protein synthesis by lymphocytes. Since the cell to cell contact between the effector cells and target cells is not possible in this assay, the reduction of colony formation should be attributed to soluble factor(s) that were secreted from the lymphocytes. Additionally, an antibody against IFN-gamma neutralized most of the antiproliferative activity, and equivalent levels of IFN-gamma were found to be present in the supernatant of PBL and LNL lower layers by a radioimmunoassay. The double layer soft agarose assay system should thus serve as a useful method for studying antiproliferative lymphokine production by lymphocytes.