Adenylate-forming enzymes can be classified into three groups: class I comprises luciferases, aryland acyl-CoA synthetases, and fatty acid-AMP ligases, while class II includes aminoacyl-tRNA synthetases required to generate charged tRNAs for protein translation via the ribosome, and class III contains adenylating enzymes found in nonribosomal peptide synthetase (NRPS)-independent siderophore (NIS) pathways. Adenylation (A) domains found in natural product biosynthetic pathways, specifically those of the nonribosomal peptide class, are part of class I and will be the focus of this review. With the class I NRPS systems, the adenylation reaction proceeds in two steps: first, adenosine triphosphate (ATP) is attached to the carboxylate, which releases inorganic pyrophosphate (PPi) and the reactive acyl-adenylate intermediate. The acyl-AMP is then transferred to a nucleophilic acceptor containing either a thiol, alcohol or amine group, generating the desired thioester, ester or amide, respectively. In NRPS assembly lines, the activated substrate reacts with the phosphotantetheine (Ppant) prosthetic group of a neighboring thiolation (T) domain or peptidyl carrier protein (PCP), which is then ready for chain extension by a condensation (C) domain. Though there does appear to be a spectrum of promiscuity that A domains exhibit, they are generally accepted as the primary gatekeepers to NRPS biosynthetic pathways and contribute to the initial structural diversity of nonribosomal peptides. Despite a lack of primary or secondary structural similarity, A domains from NRPSs are mechanistically similar to aminoacyl-tRNA synthetases from class II systems, in that they are responsible for selection and activation of the extender units in biosynthetic assembly lines. Remarkably, despite the large pool of known monomers that NRPSs can incorporate (estimated to be >300), many A domains have high substrate selectivity, and extensive work has sought to define the structural features of the binding pocket that allows for this level of molecular discrimination. This review summarizes the efforts to characterize adenylation domains from natural product NRPS pathways, with a few notable exceptions. The topics covered include the analysis of structural data and determination of substrate selectivity "codes," the variety of in vitro methods available for the biochemical characterization of A domains, and a comparison of available in silico tools for predicting A domain substrate selectivity in NRPS pathways, which has become standard practice in the postgenomic era. We will not be discussing NRPS pathway engineering strategies, which have been extensively reviewed elsewhere . Regarding the timing of substrate modifications, all A domains discussed herein load substrates that have been biosynthesized and/or modified prior to incorporation into the assembly line. We will not be addressing modifications that are installed by the NRPS during peptide extension (i.e. epimerization, cyclization, methylation, etc.), which are dictated by the embedded domain architecture, or post-assembly line tailoring reactions (i.e. methylation, halogenation, oxidoreduction, etc.), which are installed by discrete enzymes found either nearby in the gene cluster or elsewhere in the genome. The timing and enzymatic origin of these modifications in a final product can generally be discerned by careful annotation and thorough biochemical characterization of the NRPS and its gene cluster. This review covers from July 1997 up to July 2023.