We assessed the quantitative representation and specificity of alloreactive helper T lymphocytes (HTL) within murine spleen cells by three different limiting dilution systems. For the induction of primary cytolytic T lymphocyte (CTL) responses towards alloantigens, a Lyt-1+23- HTL precursor (HTLp) could be defined, which occurred at frequencies of 1/2.000-1/50,000, depending on the alloantigen in question. The HTLp limiting for interleukin-2 (Il-2) production also expressed the Lyt-1+ phenotype and occurred in similar frequencies. This cell type was concluded to be the limiting HTLp for the overall helper activity required for the induction of primary CTL responses. HTLp reactive to Mlsa -encoded antigens occurred at higher frequencies (1/500) than those reactive towards whole allogeneic H-2 haplotypes (1/4,000-1/7,000). Within the H-2 complex, I region-encoded alloantigens activated approximately 10 times more HTLp than did H-2K or H-2D regions. When alloreactive HTL were tested for antigen specificity at the clonal level, approximately 80% of the HTL clones proved to be specific to the alloantigen used for immunization, whereas approximately 20% reacted also towards third-party alloantigens. The data are discussed with respect to putative T-T interactions within the helper T cell population and the precision of alloantigen recognition by HTL.