N-terminal acetyltransferases (NATs) of bacteria acetylate the alpha-amino group in amino acids and proteins, participate in the biosynthesis of lantibiotics, and inactivate a number of antibiotics. NATs are used in biotechnology for targeted acetylation of recombinant proteins and peptides. In this regard, the search for NATs that differ in terms of substrate specificity and are also capable of functioning in the reaction at elevated temperatures, a wide pH range, etc., seems relevant. In this work, we develop specific characteristics and a search algorithm for the identification of N-terminal acetyltransferases using the Thermus thermophilus thermophilic bacterium as an example. Out of 14 Abs annotated in the genome, we selected six «putative» NATs. Some of the genes encoding the selected NATs were successfully cloned, generated, and purified from E. coli cells. The specific enzymatic activity of a number of enzymes was confirmed.