The ribonucleoprotein (RNP) machinery of the spliceosome is composed of several subunits, which assemble stepwise during the process of splicing. U2 auxiliary factor (U2AF), a heterodimer comprised of a large (65 kDa) and a small (35 kDa) subunit, is involved in the early recognition of the intron and stabilization during splicing reactions. U2AF65 specifically recognizes the polypyrimidine tract in pre-mRNA introns and additionally contacts further splicing factors, such as mBBP/SF1 and SAP155 [1,2]. U2AF65 contains three RNA recognition motifs (RRM), where RRM1 and RRM2 mediate RNA binding while RRM3 is a U2AF homology motif (UHM) that mediates the interaction with U2AF35 . A nuclear magnetic resonance spectroscopy study of recombinant human U2AF65 (RRM1-RRM2) showed a closed conformational state for the splicing factor in absence of RNA, while an open conformation is induced upon binding to polypyrimidine stretches . Here, conformational subpopulations of the protein were investigated using single-pair FRET in solution with multiparameter fluorescence detection and pulsed interleaved excitation . Information on FRET efficiency, stoichiometry, and lifetime revealed different conformational states dependent on substrate recognition and provided clues for dynamic motions of the splicing factor. These were further analyzed on a total internal reflection microscope using molecules immobilized in lipid vesicles. Changes in FRET efficiency over time showed a highly flexible U2AF65 protein with stabilization of specific conformational states upon RNA binding. Single-pair FRET measurements provide detailed insights into the mechanistic action of polypyrimidine tract recognition.