12-O-Tetradecanoylphorbol-13-acetate (TPA) inhibits DNA synthesis in bovine lymph node lymphocytes in mixed-lymphocyte cultures. Using a radioautographic study, we determined that TPA also blocked morphological changes in these cultures. Pretreatment of cultures of isologous lymphocytes with 10(-7) M TPA for three days prior to mixing was sufficient to block their subsequent response in mixed culture. Thus, TPA did not need to be present during the initial cell-to-cell interactions of the mixed lymphocyte response. The inhibition was not due to the death of the responding cell population because the effect was reversible. In one-way mixed-lymphocyte cultures, TPA pretreatment of either responding or stimulating cells could block DNA synthesis. The responding cells were more sensitive to TPA than were the stimulating cells. The inhibitory effect of the stimulators increased with an increase in the stimulating-to-responding cell ratio. In one-way cultures, it was also seen that lymphocytes from different animals varied both in their sensitivity to TPA and in their response to TPA-treated cells from other animals. The data taken together are consistent with the idea that TPA acts by changing cell surface recognition structures and/or indirectly, through activation of a subpopulation of cells to block the proliferative response. TPA may prove to be a valuable tool in studying cell-cell interactions and lymphocyte differentiation in vitro.