Properties of 2'-O-methyloligoribonucleotides containing 2'-O-(1-pyrenylmethyl)uridine were investigated as the fluorescent probe to search the single strand regions on RNA secondary and tertiary structure. The pyrene-labeled 2'-O-methyloligoribonucleotide (OMUpy) showed remarkable increase of fluorescence intensity to 333-fold at 375 nm when hybridized with the complementary oligoribonucleotide. When OMUpy, complementary to loop or stem regions, was applied to E. coli 5S-rRNA, the fluorescence intensities were increased in a sequence specific manner. The difference of the fluorescence intensities corresponds to the higher-order structure of 5S-rRNA, suggesting that pyrene-labled 2'-O-methyloligoribonucleotide can be applicable to search single strand regions of RNA.