δ-( l-α-Aminoadipyl)- l-cysteinyl- d-valine synthetase (ACVS) catalyses, via the protein thiotemplate mechanism, the nonribosomal biosynthesis of the penicillin and cephalosporin precursor tripeptide δ-( l-α-aminoadipyl)- l-cysteinyl- d-valine (ACV). The complete and fully saturated biosynthetic system approaches maximum rate of product generation with increasing ATP concentration. Nonproductive adenylation of ACVS, monitored utilising the ATP–[ 32P]PP i exchange reaction, has revealed substrate inhibition with ATP. The kinetic inhibition pattern provides evidence for the existence of a second nucleotide-binding site with possible implication in the regulatory mechanism. Under suboptimal reaction conditions, in the presence of MgATP 2−, l-Cys and inorganic pyrophosphatase, ACVS forms adenosine(5′)tetraphospho(5′)adenosine (Ap 4A) from the reverse reaction of adenylate formation involving a second ATP molecule. The potential location of the second ATP binding site was deduced from sequence comparisons and molecular visualisation in conjunction to data obtained from biochemical analysis.