The catalytic potential of tyrocidine synthetase 1 (TycA) was probed by the kinetic characterization of its adenylation activity. We observed reactions with 30 substrates, thus suggesting some substrate promiscuity. However, although the TycA adenylation (A) domain was able to accommodate alternative substrates, their k(cat)/K(M) values ranged over six orders of magnitude. A comparison of the activities allowed the systematic mapping of the substrate specificity determinants of the TycA A-domain. Hydrophobicity plays a major role in the recognition of substrate analogues but can be combined with shape complementarity, conferring higher activity, and/or steric exclusion, leading to substantial discrimination against larger substrates. A comparison of the k(cat)/K(M) values of the TycA A-domain and phenylalanyl-tRNA synthetase showed that the level of discrimination was comparable in the two enzymes for the adenylation reaction and suggested that TycA was also subjected to high selective pressure. The specificity patterns observed and the quantification of alternative activities provide a basis for exploring possible paths for the future directed evolution of A-domain specificity.