In vertebrates, carnosine synthase (EC 188.8.131.52) and carnosine N-methyltransferase (EC 184.108.40.206) are the intracellular enzymes that catalyze the formation of carnosine (β-alanyl-L-histidine) and its natural methylated derivative – anserine (β-alanyl-N-π-methyl-L-histidine), respectively. Much effort has been devoted to the elucidation of the physiological roles of these “enigmatic” imidazole dipeptides despite the still limited information available on the enzymes responsible for their biosynthesis. Carnosine synthase and chicken carnosine N-methyltransferase have only recently been molecularly identified as ATP-grasp domain containing protein 1 (ATPGD1) and histamine N-methyltransferase-like protein (HNMT-like), respectively, giving new insights into the biosynthesis of carnosine and anserine. These findings open new opportunities of research in the quest for the biological functions of imidazole dipeptides and in the assessment of their therapeutic potential. The aim of this chapter is to summarize the recent advances in our knowledge on carnosine- and anserine-producing enzymes.