Activated lymphocytes derived from tumor biopsies are very important as a source of biotherapy for cancer. The growth of lymphocytes requires periodic stimulation with specific antigen. In the case of tumor-derived lymphocytes, tumor cells in the biopsy can serve this function. This stimulation provides proliferative potential as well as functional specificity. Often, however, required numbers of viable tumor cells are not available for antigen dosing. In the present study, anti-CD3 antibody was used as a substitute for specific antigen in the generation of effector cells for biotherapy. Periodic stimulation of tumor-derived activated cells (TDAC) using anti-CD3 antibody immobilized on plastic tissue culture plates or PL-732 plastic bags resulted in continued proliferation of the TDAC. In addition, maintenance of cytotoxic activity was observed. In this study, we expanded lymphocytes from 15 tumor biopsies (five different tumor types) to therapeutic doses (greater than 5.0 X 10(10) TDAC). The average time for expansion was 70 days. Our studies showed upregulation of CD25 expression by 18 h. Proliferation of the TDAC occurred when cultures were supplemented with interleukin-2 (IL-2). The characteristics of anti-CD3 antibody-stimulated TDAC were similar to those reported before by our group using tumor biopsy for stimulation. This study provides evidence for the practical usefulness of anti-CD3 antibody stimulation of lymphocytes used in the biotherapy of cancer.