We have examined the fine specificity of a stable Thy-l.2 +, Lyt-l.2 +, Lyt-2-, and I-A s-anti-I-E k proliferating T-cell clone isolated from an A.TH anti-A.TL secondary mixed lymphocyte culture. Spleen cells from various I-A k, E k strains induced either a strong (A.TL, C3H, and CBA) or a weak (AKR and B10.BR) proliferative response, although such cells expressed at their surface similar amounts of I-E k antigens. Analysis of H-2 recombinant strains indicated that this clone recognized a conformational determinant carried by the E~ E k dimer, but not on the E k chain per se. Among the F1 hybrid strains in which the combinatorial E~ E k product was detected by cellular binding with monoclonal EL-specific antibodies (mAb), some [(B10.S(8R) x Ba0.HTT) but not others (for example, B10.A(4R)x B10.A(5R)] were stimulatory. Seventeen anti-E k mAb, regardless of the three spatially separated domains that they defined by antibody binding competition, completely inhibited the restimulation of this clone, whereas 15 other anti-A k mAb failed to do so. This clone was not reactivated by stimulating cells from strains with the H-2 haplotypes p, j, v, b, r, and s but it proliferated strongly against cells from several H-2 ~ or H-2 q strains. Genetic evidence or blocking studies with selected mAb assigned these cross-reactive mixed lymphocyte reaction determinants to the A d or A q molecules, respectively. The data support the conclusion that alloreactive T cells may define a polymorphism of/-region coded products not detected by serological analyses and extend at the T-cell level the observations of serological cross-reactions between A and E molecules.