It is generally accepted that posttranscriptional modifications in RNA play a role in the fine-tuning of RNA function and the maintenance of RNA structure. This article describes the characterization of the posttranscriptional modifications in Legionella pneumophila 16S rRNA by mass spectrometry and reverse transcriptase assays. Eight modified nucleotides were identified and mapped in the 16S rRNA sequence. Situation of these data in relation to general 16S rRNA modification patterns shows that L. pneumophila is relatively less modified, and that the majority of the L. pneumophila 16S rRNA modifications are conserved among the bacteria characterized so far (Escherichia coli. Clostridium acetobutylicum, Thermus thermophilus, and Thermotoga maritima).