The identification of benzimidazole incorporated into RNA of Escherichia coli as benzimidazole nucleoside by means of mass spectrometry is reported. Trimethylsilylation of an enzymatic digest of bacterial RNA allowed the separation of the different nucleosides by gas chromatography. The coupled mass spectrometer was used as a mass specific detector and allowed the sensitive detection of single components of the complex mixture. Thus, benzimidazole ribonucleoside could be detected in hydrolysates of RNA from E. coli fed benzimidazole in the culture broth, although this nucleoside could not be completely separated from uridine by the gas chromatographic systems explored. Quantitation of the benzimidazole nucleoside content revealed that benzimidazole is incorporated into RNA amounting to 16% relative to adenosine.