The pseuoduridine synthases (psi synthases) isomerize uridine (U) to pseudouridine (psi) in RNA, and they fall into five families that share very limited sequence similarity but have the same overall fold and active-site architecture, including an essential Asp. The mechanism by which the psi synthases operate remains unknown, and mechanistic work has largely made use of RNA containing 5-fluorouridine (f5U) in place of U. The psi synthase TruA forms a covalent adduct with such RNA, and heat disruption of the adduct generates a hydrated product of f5U, which was reasonably concluded to result from the hydrolysis of an ester linkage between the essential Asp and f5U. In contrast, the psi synthase TruB, which is a member of a different family, does not form an adduct with f5U in RNA but catalyzes the rearrangement and hydration of the f5U, which labeling studies with [18O]water showed does not result from ester hydrolysis. To extend the line of mechanistic investigation to another family of psi synthases and an enzyme that makes an adduct with f5U in RNA, the behavior of RluA toward RNA containing f5U was examined. Stem-loop RNAs are shown to be good substrates for RluA. Heat denaturation of the adduct between RluA and RNA containing f5U produces a hydrated nucleoside product, and labeling studies show that hydration does not occur by ester hydrolysis. These results are interpreted in light of a consistent mechanistic scheme for the handling of f5U by psi synthases.