The antigen-induced proliferative response of lymph node cells (LNC) from mice sensitized to the monofunctional antigen L-tyrosine-p-azobenzenearsonate (ABA-Tyr) was used to monitor genetic control. All strains tested mounted significant responses, but those that were H-2(b) at both the I-A and I-E loci [B10., B6., B10.A(18R), A.BY, and C3H.SW] gave consistently weaker responses than other haplotypes. The F(1) progeny of matings between high and low responder phenotype parents (DBA/2 and B6, respectively) were high responders, establishing the dominance of the responder trait. Proliferative responses of LNC to ABA-Tyr were blocked by the appropriate anti-Ia monoclonal reagents. For example, B10.A(4R) LNCI (I-A(k), I-E(b)) were blocked by anti-I-A(k), whereas B10.A(3R) LNC (I-A(b), I-E(k)) were blocked by anti-I-E(k). Long-term cultures of T cell lines specifically reactive to ABA-Tyr were established from LNC of A/J mice immunized with ABA-Tyr and were cloned by limiting dilution. The proliferative responses to ABA-Tyr of 14 out of 15 clones tested were I-A restricted on the basis of activation by antigen-presenting cells from appropriate recombinant strains and the blocking activity of the monoclonal anti-Ia antibodies. The response of the other clone was I-E restricted. The fine antigen specificity of the clones was studied using structural analogs of the homologous antigen to induce proliferation. The clones could be divided into three types with respect to responsiveness to ABA-histidine (ABA-His). One group responded about equally well to ABA-His and ABA-Tyr. A second set responded less strongly to ABA-His than to ABA-Tyr, while the third showed no response above background to ABA- His. In all instances, the ABA-His-responding clones discriminated exquisitely between the 2-azo and 4-azo histidine isomers, responding only to the 4-azo compound. These T cell clones provide extremely useful tools for studies of T cell specificity, antigen recognition and lymphoid cell interaction systems.