We have studied the properties of helper T cells specific for sheep erythrocytes (SRBC), keyhole limpet hemocyanin (KLH), or poly-L-(Tyr,Glu)-poly-DL-Ala-poly-L-Lys [(T,G)-A--L]. These T cells differentiated and were primed in vivo in irradiation chimeras constructed of various combinations of F1 and parental bone marrow donors and irradiated recipients. Primed T cells were then tested for helper activity in the in vitro response of B cells and macrophages (Mphi) of parental or F1 origin to the hapten trinitrophenol coupled to the priming antigen. When testing either SRBC or KLH-specific T cells of parental H-2 type which had differentiated in F1 hosts, we found that they cooperated equally well with B cells and Mphi of either parental H-2 type. On the other hand, when testing F1 T cells which had differentiated in parental hosts, we found that they cooperated well only with B cells and Mphi which had the K-IA region type of the parental host. In similar experiments we found that (T,G)-A--L-specific T cells of low responder H-2 type which had differentiated in (high responder X low responder) F1 hosts induced high responses in high responder B cells and Mphi (T,G)-A--L-specific F1 T cells which differentiated in high responder but not those which differentiated in low responder hosts induced high responses in high responder B cells and Mphi. Low responder B cells and Mphi yielded low responses in all cases regardless of the source of (T,G)-A--L-specific T cells with what they were tested. Our results support the conclusion that I-region and Ir genes function via their expression in B cells and Mphi and in the host environment during helper T-cell differentiation, but not, at least under the conditions of these experiments, via their expression in the helper T cell itself. These findings place constraints upon models which attempt to explain the apparent dual recognition of antigen and I-region gene products by helper T cells.