Specific selection of antigen-responsive guinea pig peritoneal exudate lymphocytes (PELs) was achieved by a selection culture procedure. This procedure involved the addition of PELs from immune donors to monolayers of antigen-pulsed adherent peritoneal exudate cells from nonprimed syngeneic donors. PELs which failed to adhere were discarded at 24 and 48 h; after 1 wk of culture, lymphocytes were obtained which were highly responsive to the antigen for which they were selected but which demonstrated little or no response to other antigens to which the original donor of the lymphocyte was immune. These selected cells were largely T lymphocytes and could be maintained in culture for 2-5 wk in an antigen-responsive state and, in 20-30% of cases, for 8-10 wk in an antigen-independent state.