Effector mechanisms in experimental autoimmune thyroiditis (EAT) were studied in vitro by establishing a cytotoxicity system with thyroid target cells. Lymph node cells (LNC) from popliteal and inguinal lymph nodes were obtained from CBA/J mice (8-10 wk old) 12-18 d after immunization with 120 micrograms mouse thyroglobulin (MTg) in complete Freund's adjuvant (0.2 ml to both hind footpads and thighs) and were cultured with MTg (10-50 micrograms/ml). On day 5 of culture, viable LNC were added to labeled thyroid monolayers and their cytoxicity was assayed after 16 h. Functional thyroid target cells, as reflected by MTg production for up to 9 d, were prepared by adding 1 mM dibutyryl adenosine 3',5'-cyclic monophosphate and 60 microU thyroid-stimulating hormone/ml to the culture medium. On days 5-7, confluent monolayers were labeled with 111In and used as targets. Specific 111In-release ranged from 56 to 85%. The cytotoxic response is MTg specific and H-2 restricted. Pretreatment of thyroid target cells with rabbit antiserum to MTg completely inhibited cytotoxicity. Pretreatment with mouse antiserum to either Kk or Dk products resulted in approximately 50% inhibition, whereas the combined use of both antisera led to total inhibition. No cytotoxicity was observed when control BALB/c thyroid cultures were the target cells. The kinetics of the expansion of Thy-1+ cytotoxic cells by in vitro exposure to MTg were then studied. The cytotoxic response required 5 d to develop and was abolished by treating LNC on day 4 with monoclonal antibody to Lyt-1.1, but not to Lyt-2.1, plus complement. In contrast, by day 5, cytotoxicity was abrogated by similar treatment with antiserum to Lyt-2.1, but not to Lyt-1.1. We conclude that cytotoxic cells derived from MTg-immunized mice are Lyt-2-bearing cells but require the presence of Lyt-1-bearing cells for their generation and/or differentiation.