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Pseudouridine (5-β-d-ribofuranosyluracil, Ψ) is the most commonly found modified base in RNA. Conversion of uridine to Ψ is performed enzymatically in both prokaryotes and eukaryotes by pseudouridine synthases (EC 4.2.1.70). The Escherichia coli Ψ-synthase RluD modifies uridine to Ψ at positions 1911, 1915 and 1917 within 23 S rRNA. RluD also possesses a second function related to proper assembly of the 50 S ribosomal subunit that is independent of Ψ-synthesis. Here, we report the crystal structure of the catalytic module of RluD (residues 68–326; ΔRluD) refined at 1.8 A˚ to a final R-factor of 21.8% (Rfree=24.3%). ΔRluD is a monomeric enzyme having an overall mixed α/β fold. The ΔRluD molecule consists of two subdomains, a catalytic subdomain and C-terminal subdomain with the RNA-binding cleft formed by loops extending from the catalytic sub-domain. The catalytic sub-domain of ΔRluD has a similar fold as in TruA, TruB and RsuA, with the location of the RNA-binding cleft, active-site and conserved, catalytic Asp residue superposing in all four structures. Superposition of the crystal structure of TruB bound to a T-stem loop with RluD reveals that similar RNA–protein interactions for the flipped-out uridine base would exist in both structures, implying that base-flipping is necessary for catalysis. This observation also implies that the specificity determinants for site-specific RNA-binding and recognition likely reside in parts of RluD beyond the active site. [Copyright &y& Elsevier]