Structure-specific nucleases called 5' flap endonucleases cleave unannealed 5' arms of template-primer DNA model substrates at the start of the duplex and are involved in Okazaki fragment processing during DNA synthesis. To determine the possible use of the enzymes in RNA structure analysis, the cleavage of synthetic and native RNAs was examined using flap endonuclease 1 (Fen1) of HeLa cells. RNAs are cleaved at about 20% of the rate of DNA model substrates, and most of the cleavage sites are within 200 nucleotides of the 5' end. Hydrolysis of MFA2 mRNA of yeast shows that the cleavages are at the start of five possible stem structures of a folded secondary structure predicted on the basis of both chemical and enzymatic structure probing. 16S ribosomal RNA of Escherichia coli is cleaved at several 5' stem structures of its phylogenetically predicted folded structure. This type of RNA cleavage specificity may be very useful in secondary structure analysis in the future and also may be used by cells for specific 5' end-geared RNA cleavages.