The large subunit of the human U2 small nuclear ribonucleoprotein particle auxiliary factor (hU2AF(65)) is an essential RNA-splicing factor required for the recognition of the polypyrimidine tract immediately upstream of the 3' splice site. In the present study, we determined the solution structures of two hU2AF(65) fragments, corresponding to the first and second RNA-binding domains (RBD1 and RBD2, respectively), by nuclear magnetic resonance spectroscopy. The tertiary structure of RBD2 is similar to that of typical RNA-binding domains with the beta1-alpha1-beta2-beta3-alpha2-beta4 topology. In contrast, the hU2AF(65) RBD1 structure has unique features: (i) the alpha1 helix is elongated by one turn toward the C-terminus; (ii) the loop between alpha1 and beta2 (the alpha1/beta2 loop) is much longer and has a defined conformation; (iii) the beta2 strand is (188)AVQIN(192), which was not predicted by sequence alignments; and (iv) the beta2/beta3 loop is much shorter. Chemical shift perturbation experiments showed that the U2AF-binding RNA fragments interact with the four beta-strands of RBD2 whereas, in contrast, they interact with beta1, beta3 and beta4, but not with beta2 or the alpha1/beta2 loop, of RBD1. The characteristic alpha1-beta2 structure of the hU2AF(65) RBD1 may interact with other proteins, such as UAP56.