Mouse IL-12 acts on both mouse and human cells; human IL-12 acts only on human cells. This species specificity is determined by the p35 subunit of the IL-12 heterodimer. Since mouse and human p35 sequences are 60% identical, the determinants for the species specificity most likely residue in the nonhomologous sequences of mouse p35. To identify the regions on the p35 subunit interacting with the mouse IL-12 receptor, we constructed a series of chimeric mouse-human p35 molecules by replacing mouse sequences with the nonhomologous human counterparts. An IL-12 heterodimer containing a mouse-human p35 chimera with five residues changed in three discontinuous sites had drastically reduced (750-3000-fold) bioactivities on mouse cells. However, the competitive binding activity of the same mutant IL-12 heterodimer on mouse cells was only reduced 30-fold relative to wild-type IL-12. These findings therefore suggest that 1) the mouse p35 subunit participates in both receptor binding and signaling, 2) the mutations introduced into p35 affect signaling to a much greater extent than receptor binding, and 3) the five residues identified on p35 are required for interacting with the mouse, but not with the human IL-12 receptor and as such contribute extensively to the observed species specificity of IL-12.