Previous work indicates that aminoacyl-tRNA synthetases make a transient covalent adduct with cognate tRNAs, through Michael addition of an enzyme nucleophile to the carbon-6 position of uridine 8. We report the selective reduction of the 5,6 double bond of 4-thiouridine at position 8 in Escherichia coli tyrosine tRNA, so as to prevent formation of the presumed covalent enzyme-nucleic acid adduct. The completely reduced tRNA molecules are inactivated for aminoacylation. With partial reduction, a mixed pool of active and inactive molecules is created and the degree of inactivation exactly matches the extent of 4-thiouridine reduction. The active molecules recovered from this mixed pool are specifically unaltered at position 8. The results are consistent with the view that the covalent enzyme-RNA adduct is an obligatory intermediate for aminoacylation of this tRNA.