A cDNA encoding mouse pseudouridine synthase 3 (mPus3p) has been cloned. The predicted protein has 34% identity with yeast pseudouridine synthase 3 (Pus3), an enzyme known to form pseudouridine at positions 38 and 39 in yeast tRNA. The cDNA is 1.7 kb, and when used as a probe on a Northern blot of total RNA from mouse tissues or cells in culture, a band at 1.8 kb was observed. The open reading frame codes for a protein of 481 amino acids with a predicted molecular mass of 55 552 Da. When mPus3p was in vitro translated and used in reactions with tRNA substrates from both yeast and humans, uridines at position 39 were modified to pseudouridine. In a tRNA substrate with a uridine at position 38 (human tRNA(Leu)), there was very slight formation of pseudouridine at that position after incubation with mPus3p.