ASPP2 is a pro-apoptotic protein that stimulates the p53-mediated apoptotic response (1). The C-terminal Ankyrin and SH3 domains in ASPP2 mediate numerous protein-protein interactions. In addition, ASPP2 contains a proline-rich domain between residues 693-918, whose structure and function are unclear (2). Here we show using biophysical methods that the proline-rich domain of ASPP2 is natively unfolded. CD spectroscopy gave a spectrum typical for a random coil, which did not change upon increasing the temperature. Analytical ultracentrifugation studies showed that the protein is a monomer. However, in analytical gel filtration experiments the protein was eluted earlier than expected for globular protein. The results of different computational predictions of ASPP2 693-918 also revealed that the domain is mostly natively unfolded. Next, we studied the role of this domain in protein-protein interactions. Using peptide arrays bearing overlapping peptides derived from known ASPP2 binding proteins we demonstrate that only the Ank-SH3 domains, but not the proline-rich domain, mediate the interactions of ASPP2 with its partner proteins. Instead, we found using fluorescence spectroscopy and pull-down binding studies that the proline-rich domain makes an intramolecular interaction with the Ank-SH3 domain of ASPP2. We have mapped the precise sites that mediate this intramolecular domain-domain interaction using a second peptide array. According to our model, the intramolecular interaction between the ASPP2 domains has an important role in regulating the intermolecular interactions of ASPP2 with its partner proteins. When the proline-rich domain is bound to the Ank-SH3 domains, it competitively inhibits their interactions with their partner proteins. Following a yet unknown regulation mechanism, the intramolecular domain-domain interaction is released and the Ank-SH3 domains become available to bind their target proteins.