We have utilized a clonal approach to investigate functional and immunogenetic characteristics of T cells responsive to the miHA H-1.3. Our data prove the existence of H-2 I region-restricted helper T cells that are specific for H-1.3, or for determinants encoded by closely linked loci. These conclusions are based upon the following observations: 1) the cloned Th are driven to proliferate only by H-2 I-compatible stimulator cells that bear the appropriate (H-1.3) miHA; 2) antigenic stimulation causes the cloned Th to secrete lymphokine with high levels of IL 2-like activity; and 3) they are not specifically lytic for relevant target cells. These cloned Th were isolated at a rather low frequency from an H-1.3-immune MLC population compared with the observed frequency of H-1.3-specific Tc that proliferate autonomously after antigen stimulation; i.e., HITc isolated from the same MLC. They were, however, capable of promoting the clonal expansion of an H-1.3-specific HITc, suggesting that Th can collaborate with HITc in an anti-miHA response.