The function of accessory cells in primary and secondary in vitro antibody responses to TNP-(T,G)-A--L and TNP-(H,G)-A--L is under the control of immune response (Ir) genes which map to I-A. The expression of Ir gene function by B cells is related to the B cell activation pathway; Ir gene function is expressed by B cells activated under conditions involving MHC-restricted T-B interaction. In vitro augmented primary and secondary responses to TNP-nuclease (TNP-NASE) have also been established and documented to be under the control of H-2 linked Ir gene(s) mapping to the I-B subregion. For these responses, accessory cell function was shown to be under Ir gene control. Recent data employing monoclonal anti-Ia reagents have suggested that genes in the I-A subregion may also be involved in regulating responses to TNP-NASE. In order to further analyze the genetic regulation of T cell responses to NASE, a series of cloned lines were generated in BALB/c (H-2d) as well as (H-2b x H-2a)F1 T cells. Individual clones were restricted to recognizing NASE in the context of either AAlphaABeta or EAlphaEBeta products. The antigen fine specificity of cloned NASE-specific T cells was also probed through the use of mutant NASE molecules and synthetic peptides corresponding to segments of NASE. A consistent correlation was found between the fine specificity of a given clone and its MHC restriction specificity. AAlphab ABetab restricted clones were selectively responsive to peptide 91-110; EAlphak EBetak restricted clones were responsive to peptide 81-100.