Cells have developed molecular machineries, which can chemically modify DNA and RNA nucleosides. One particular and chemically simple modification, (cytosine-5) methylation (m(5)C), has been detected both in RNA and DNA suggesting universal use of m(5)C for the function of these nucleotide polymers. m(5)C can be reproducibly mapped to abundant noncoding RNAs (transfer RNA, tRNA and ribosomal RNA, rRNA), and recently, also nonabundant RNAs (including mRNAs) have been reported to carry this modification. Quantification of m(5)C content in total RNA preparations indicates that a limited number of RNAs carry this modification and suggests specific functions for (cytosine-5) RNA methylation. What exactly is the biological function of m(5)C in RNA? Before attempting to address this question, m(5)C needs to be mapped specifically and reproducibly, preferably on a transcriptome-wide scale. To facilitate the detection of m(5)C in its sequence context, RNA bisulfite sequencing (RNA-BisSeq) has been developed. This method relies on the efficient chemical deamination of nonmethylated cytosine, which can be read out as single nucleotide polymorphism (nonmethylated cytosine as thymine vs. methylated cytosine as cytosine), when differentially comparing cDNA libraries to reference sequences after DNA sequencing. Here, the basic protocol of RNA-BisSeq, its current applications and limitations are described.