RNA 3'-terminal phosphate cyclases are a family of evolutionarily conserved enzymes that catalyze ATP-dependent conversion of the 3'-phosphate to the 2',3'-cyclic phosphodiester at the end of RNA. The precise function of cyclases is not known, but they may be responsible for generating or regenerating cyclic phosphate RNA ends required by eukaryotic and prokaryotic RNA ligases. Previous work carried out with human and Escherichia coli enzymes demonstrated that the initial step of the cyclization reaction involves adenylation of the protein. The AMP group is then transferred to the 3'-phosphate in RNA, yielding an RNA-N(3')pp(5')A (N is any nucleoside) intermediate, which finally undergoes cyclization. In this work, by using different protease digestions and mass spectrometry, we assign the site of adenylation in the E. coli cyclase to His-309. This histidine is conserved in all members of the class I subfamily of cyclases identified by phylogenetic analysis. Replacement of His-309 with asparagine or alanine abrogates both enzyme-adenylate formation and cyclization of the 3'-terminal phosphate in a model RNA substrate. The cyclase is the only known protein undergoing adenylation on a histidine residue. Sequences flanking the adenylated histidine in cyclases do not resemble those found in other proteins modified by nucleotidylation.