Lgt1 is one of the glucosyltransferases produced by the Gram-negative bacterium Legionella pneumophila. This enzyme modifies eukaryotic elongation factor 1A (eEF1A) at serine 53, which leads to inhibition of protein synthesis and death of target cells. Here we studied the region of eEF1A, which is essential for substrate recognition by Lgt1. We report that the decapeptide (50)GKGSFKYAWV(59) of eEF1A is efficiently modified by Lgt1. This peptide covers the loop of the helix-loop-helix region formed by helices A* and A' of eEF1A and is part of the first turn of helix A'. Substitution of either serine 53, phenylalanine 54, tyrosine 56, or tryptophan 58 by alanine abolished or severely decreased glucosylation. Lgt1 modified the decapeptide (50)GKGSFKYAWV(59) with a higher glucosylation rate than full-length eEF1A purified from yeast, suggesting that a specific conformation of eEF1A is the preferred substrate of Lgt1. A GenBank search on the basis of the substrate decapeptide for similar peptide sequences retrieved heat shock protein 70 subfamily B suppressor 1 (Hbs1) as a target for glucosylation by Lgt1. Recombinant Hbs1 and the corresponding fragment ((303)GKASFAYAWV(312)) were gluco syl a ted by Lgt1. NMR studies with the gluco syl a ted eEF1A-derived decapeptide identified an alpha-anomeric structure of the glucose-serine 53 bond and characterize Lgt1 as a retaining glucosyltransferase.