A Bacillus subtilis ORF, ypul, 41% homologous to rsuA, the gene for the synthase which forms pseudouridine 516 in Escherichia coli 16S rRNA, was cloned and the protein expressed and affinity-purified by the His tag procedure. Reactions with E. coli 16S and 23S rRNA transcripts were performed in vitro. The protein did not form pseudouridine 516 as expected but did produce pseudouridine 552 in 16S rRNA and pseudouridines 1199, 2605, and 2833 in 23S rRNA. Of these, only pseudouridine 2605 is found naturally in either E. coli or B. subtilis rRNA. Kinetic experiments confirmed that pseudouridine 2605 was the primary target. Comparison of the four pseudouridine sites yielded a consensus recognition sequence for the synthase. This consensus sequence was not present at any other site in either E. coli or B. subtilis 16S or 23S RNA. We propose that YpuL is the B. subtilis pseudouridine 2633 (2605 in E. coli) synthase. Since the closest gene sequence homologue in E. coli is yciL, we suggest that its gene product is the corresponding E. coli pseudouridine 2605 synthase.