Human Nck2 (hNck2) is a 380-residue adapter protein consisting of three SH3 domains and one SH2 domain. Nck2 plays a pivotal role in connecting and integrating signaling networks constituted by transmembrane receptors such as ephrinB and effectors critical for cytoskeletonal dynamics and remodeling. In this study, we aimed to determine the NMR structures and dynamic properties of the hNck2 SH3 domains and to define their ligand binding preferences with nine proline-rich peptides derived from Wire, CAP-1, CAP-2, Prk, Wrch1, Wrch2, and Nogo. The results indicate (1) the first hNck2 SH3 domain is totally insoluble. On the other hand, although the second and third hNck2 SH3 domains adopt a conserved SH3 fold, they exhibit distinctive dynamic properties. Interestingly, the third SH3 domain has a far-UV CD spectrum typical of a largely unstructured protein but exhibits {1H}-15N steady-state NOE values larger than 0.7 for most residues. (2) The HSQC titrations revealed that the two SH3 domains have differential ligand preferences. The second SH3 domain seems to prefer a consensus sequence of APx#PxR, while the third SH3 domain prefers PxAPxR. (3) Several high-affinity bindings were identified for hNck2 SH3 domains by isothermal titration calorimetry. In particular, the binding of SH3-3 with the Nogo-A peptide was discovered and shown to exhibit a Kd of 5.7 microM. Interestingly, of the three SH3-binding motifs carried by Wrch1, only the middle one was capable of binding SH3-2. Our results provide valuable clues for further functional investigations into the Nck2-mediated signaling networks.