Transforming growth factor β (TGF-β) is secreted primarily as a latent complex consisting of the TGF-β homodimer, the TGF-β propeptides (called the latency-associated protein or LAP) and the latent TGF-β binding protein (LTBP). Mature TGF-β remains associated with LAP by non-covalent interactions that block TGF-β from binding to its receptor. Complex formation between LAP and LTBP is mediated by an intramolecular disulfide exchange between the third 8-cysteine (8-Cys3) domain of LTBP with a pair of cysteine residues in LAP. Only the third 8-Cys domains of LTBP-1, -3, and -4 bind LAP. From comparison of the 8-Cys3_LTBP-1 structure with that of the non-TGF-β-binding 8-Cys6_fibrillin-1, we observed that a two-residue insertion in 8-Cys3_LTBP-1 increased the potential for disulfide exchange of the 2–6 disulfide bond. We further proposed that five negatively charged amino acid residues surrounding this bond mediate initial protein–protein association. To validate this hypothesis, we monitored binding by fluorescence resonance energy transfer (FRET) analysis and co-expression assays with TGF-β1 LAP (LAP-1) and wild-type and mutant 8-Cys3 domains. FRET experiments demonstrated ionic interactions between LAP-1 and 8-Cys3. Mutation of the five amino acid residues revealed that efficient complex formation is most dependent on two of these residues. Although 8-Cys3_LTBP-1 binds proTGF-βs effectively, the domain from LTBP-4 does so poorly. We speculated that this difference was due to the substitution of three acidic residues by alanine, serine, and arginine in the LTBP-4 sequence. Additional experiments with 8-Cys3_LTBP-4 indicated that enhanced binding of LAP to 8-Cys3_LTBP-4 is achieved if the residues A, S, and R are changed to those in 8-Cys3_LTBP1 (D, D, and E) and the QQ dipeptide insertion of LTBP-4 is changed to the FP in 8-Cys3_LTBP-1. These studies identify surface residues that contribute to the interactions of 8-Cys3 and LAP-1 and may yield information germane to the interaction of 8-Cys domains and additional TGF-β superfamily propeptides, an emerging paradigm for growth factor regulation.