DNA synthesis (3H-thymidine incorporation) of DA rat spleen and lymph node cells cultured with PHA or (DA x Lewis)F1 lymph node cells was profoundly inhibited by pretreatment with large i.v. doses of antigen (greater than 100 mg OA or greater than 10(10) SRBC) 24 hr before culture. This effect, seen as early as 3 to 6 hr after antigen administration, had disappeared within 7 days. Similar depressed spleen cell responses were seen 9 days after footpad injection of 200 mug OA in complete adjuvant. Lymph node responses showed a similar inhibition only in the presence of specific antigen in culture. In all experiments in which antigen administration resulted in decreased responses, removal of glass wool adherent cells restored responsiveness to normal. The failure of purified macrophages to reestablish inhibition favored the interpretation that an adherent suppressor cell distinct from the macrophage is activated by antigen in vivo to regulate T cell responses in vitro.