Through the use of various non-equilibrium RNA binding techniques, the C protein tetramer of mammalian 40S hnRNP particles has been characterized previously as a poly(U) binding protein with specificity for the pyrimidine-rich sequences that often precede 3′ intron-exon junctions. C protein has also been characterized as a sequence-independent RNA chaperonin that is distributed along nascent transcripts through cooperative binding and as a protein ruler that defines the length of RNA packaged in 40S monoparticles. In this study fluorescence spectroscopy was used to monitor C protein-oligonucleotide binding in a competition binding assay under equilibrium conditions. Twenty nucleotide substrates corresponding to polypyrimidine tracts from IVS1 of the adenovirus-2 major late transcript, the adenovirus-2 oncoprotein E1A 3′ splice site, IVS2 of human α-tropomyosin, the consensus polypyrimidine tract for U2AF65, AUUUA repeats and r(U)20 were used as competitors. A 20 nt β-globin intronic sequence and a randomly generated oligo were used as competitor controls. These studies reveal that native C protein possesses no enhanced affinity for uridine-rich oligonucleotides, but they confirm the enhanced affinity of C protein for an oligonucleotide identified as a high affinity substrate through selection and amplification. Evidence that the affinity of C protein for the winner sequence is due primarily to its unique structure or to a unique context is seen in its retained substrate affinity when contiguous uridines are replaced with contiguous guanosines.