We have compared various kinetic and melting properties of oligoribonucleotide probes containing 2'-O-methylnucleotides or 2'-deoxynucleotides with regard to their use in assays for the detection of nucleic acid targets. 2'-O-Methyl oligoribonucleotide probes bound to RNA targets faster and with much higher melting temperatures (Tm values) than corresponding 2'-deoxy oligoribonucleotide probes at all lengths tested (8-26 bases). Tm values of both probes increased with length up to approximately 19 bases, with maximal differences in Tm between 2'-O-methyl and 2'-deoxy oligoribonucleotide probes observed at lengths of 16 bases or less. In contrast to RNA targets, 2'-O-methyl oligoribonucleotide probes bound more slowly and with the same Tm to DNA targets as corresponding 2'-deoxy oligoribonucleotide probes. Because of their greatly enhanced Tm when bound to RNA, 2'-O-methyl oligoribonucleotide probes can efficiently bind to double-stranded regions of structured RNA molecules. A 17 base 2'-O-methyl oligoribonucleotide probe was able to bind a double-stranded region of rRNA whereas the same 17 base 2'- deoxy oligoribonucleotide probe did not. Due to their enhanced Tm when bound to RNA targets, shorter 2'-O-methyl oligoribonucleotide probes can be used in assays in place of longer 2'-deoxy oligoribonucleotide probes, resulting in enhanced discrimination between matched and mismatched RNA targets. A 12 base 2'-O-methyl oligoribonucleotide probe had the same Tm as a 19 base 2'-deoxy oligoribonucleotide probe when bound to a matched RNA target but exhibited a much larger decrease in Tm than the 2'-deoxy oligoribonucleotide probe when bound to an RNA target containing either 1 or 2 mismatched bases. The increased Tm, faster kinetics of hybridization, ability to bind to structured targets and increased specificity of 2'-O-methyl oligoribonucleotide probes render them superior to corresponding 2'-deoxy oligoribonucleotides for use in assays that detect RNA targets.