Proline-rich motifs are widely distributed in eukaryotic proteomes and are usually involved in the assembly of functional complexes through interaction with specific binding modules. The tumour-suppressor p53 protein presents a proline-rich region that is crucial for regulating apoptosis by connecting the p53 with a complex protein network. In humans, a common polymorphism determines the identity of residue 72, either proline or arginine, and affects the features of the motifs present in the polyproline domain. The two isoforms have different biochemical properties and markedly influence cancer onset and progression. In this article, we analyse the binding of the p53 proline-rich region with a pool of selected polyproline binding domains (i.e. SH3 and WW), and we present the first demonstration that the purified SH3 domains of the CD2AP/Cin85 protein family are able to directly bind the p53 protein, and to discriminate between the two polymorphic variants P72R.