We have synthesized the analogue 2′-deoxy-2′-thio-CTP (CTP-SH) and tested its ability to support RNA transcription in place of CTP. The modified nucleotide in a transcription reaction and in the absence of CTP generated the appropriately sized fragment when a mutant T7 polymerase (Y639F) was used. Wild-type polymerase was unable to generate RNA under the same conditions. Transcription was optimal around pH 7.5 and was dependent upon CTP-SH concentration. Transcripts containing the analogue were efficiently isolated using a thiol-activated sepharose column. Insertion of CTP-SH into the HDV ribozyme, replacing all cytidine residues with 2′-thiocytidine, appears to inhibit self-cleaving activity, even in the presence of manganese. The ability to introduce the CTP-SH analogue enzymatically into RNA opens the way for new structure–function studies where the 2′-hydroxyl can be efficiently replaced by a thiol group.