Pseudouridine is the most abundant of more than 100 chemically distinct natural ribonucleotide modifications. Its synthesis consists of an isomerization reaction of a uridine residue in the RNA chain and is catalyzed by pseudouridine synthases. The unusual reaction mechanism has become the object of renewed research effort, frequently involving replacement of the substrate uridines with 5-fluorouracil (f(5)U). f(5)U is known to be a potent inhibitor of pseudouridine synthase activity, but the effect varies among the target pseudouridine synthases. Derivatives of f(5)U have previously been detected, which are thought to be either hydrolysis products of covalent enzyme-RNA adducts, or isomerization intermediates. Here we describe the interaction of pseudouridine synthase 1 (Pus1p) with f(5)U-containing tRNA. The interaction described is specific to Pus1p and position 27 in the tRNA anticodon stem, but the enzyme neither forms a covalent adduct nor stalls at a previously identified reaction intermediate of f(5)U. The f(5)U27 residue, as analyzed by a DNAzyme-based assay using TLC and mass spectrometry, displayed physicochemical properties unaltered by the reversible interaction with Pus1p. Thus, Pus1p binds an f(5)U-containing substrate, but, in contrast to other pseudouridine synthases, leaves the chemical structure of f(5)U unchanged. The specific, but nonproductive, interaction demonstrated here thus constitutes an intermediate of Pus turnover, stalled by the presence of f(5)U in an early state of catalysis. Observation of the interaction of Pus1p with fluorescence-labeled tRNA by a real-time readout of fluorescence anisotropy and FRET revealed significant structural distortion of f(5)U-tRNA structure in the stalled intermediate state of pseudouridine catalysis.