RlmH is a S-adenosylmethionine-dependent, homodimeric SPOUT methyltransferase that methylates selectively the N-3 position of pseudouridine 1915 to form m3Y1915 in the loop of helix 69 (Domain IV) of 23S rRNA. RlmH protein is able to methylate pseudouridine in vitro using only 70S ribosomes, but not 50S subunits from the ybeA deletion strain as substrate. It preferentially methylates Y1915 and less efficiently U1915, but not Y at position 1911 or 1917. It docks comfortably into the ribosomal A site without encroaching into the P site. Homologs of E. coli RlmH are present in most bacterial lineages, but are essentially absent in the Archaea and Eukaryota. Formation of Y1915 is dependent on the gene product of rluD.