The Sin3 co-repressor acts as a protein scaffold to recruit transcription factors via its four highly homologous paired amphipathic helix (PAH) domains. PAH2 has been shown to interact strongly with the Sin3 interacting domain (SID) of the tumor suppressor Mad. This PAH2/Mad complex has been studied extensively by NMR, but the molecular determinants that dictate the specificity of interaction remain to be elucidated. To uncover residues that convey the specificity of interaction between PAH2 and Mad, PAH2 residues contacted by the Mad-SID were introduced into the PAH1 domain of mSin3b and tested for gain-of-interaction in vivo in a yeast two-hybrid setting and further confirmed in a cell-free system. This approach led to the identification of PAH2-Phe-7 as a critical residue. Stabilization of the interaction between PAH1-Phe-7 and the Mad-SID was achieved by introducing Val-14 and Gln-39 into PAH1. Substitution of PAH2 residues contacted by the Mad-SID with their respective residues in PAH1 corroborated and extended the critical role of Phe-7 and the stabilizing role of Val-14 and Gln-39. We conclude that Phe-7 is the critical determinant and provides the molecular specificity for the association between Sin3 and Mad in regulating cell growth and differentiation.