Aminoacyl-tRNAs were long thought to be involved solely in ribosome dependent protein synthesis and essential primary metabolism processes, such as targeted protein degradation and peptidoglycan synthesis. About 10 years ago, an aminoacyl-tRNA-dependent enzyme involved in the biosynthesis of the antibiotic valanimycin was discovered in a Streptomyces strain. Far from being an isolated case, this discovery has been followed by the description of an increasing number of aminoacyl-tRNA-dependent enzymes involved in secondary metabolism. This review describes the three groups of aminoacyl-tRNA-dependent enzymes involved in the synthesis of natural products. Each group is characterized by a particular chemical reaction, and its members are predicted to share a specific fold. The three groups are cyclodipeptide synthases involved in diketopiperazine synthesis, LanB-like dehydratases involved in the posttranslational modification of ribosomal peptides, and transferases from various biosynthesis pathways.