The mechanisms responsible for the induction of I-J restrictions on third-order suppressor T cells (TS3) were analyzed. The I-J phenotype of the antigen-coupled cells used for priming restricted the specificity of the TS3 population. Thus, TS3 cells were only generated after priming with antigen-coupled I-J homologous cells. Identity at the I-JM (and I-E) subregions was sufficient for TS3 induction. Furthermore, priming of H-2 heterozygous mice with antigen-coupled parental cells generated TS3 that were restricted to the parental haplotype used for priming. The splenic cell population responsible for antigen presentation and induction of TS3 cells was fractionated. The cells involved in antigen presentation were found in the splenic adherent population and were absent in the fraction containing splenic nonadherent T and B cells. The subsequent activation and interaction of TS3 cells is also restricted by genes in the H-2 complex. The results are discussed in terms of a general mechanism responsible for the induction of restrictions in T helper and TS3 cells.