The initial step of tryptophan biosynthesis is catalyzed by the enzyme anthranilate synthase, which in most microorganisms is subject to feedback inhibition by the end product of the pathway. We have characterized the TRP2 gene from a mutant Saccharomyces cerevisiae strain coding for an anthranilate synthase that is unresponsive to tryptophan. Sequence analysis of this TRP2(Fbr) (feedback-resistant) allele revealed numerous differences from a previously published TRP2 sequence. However, TRP2(Fbr) was found to differ in only one single-point mutation from its own parent wild type, a C-to-T transition resulting in a serine 76-to-leucine 76 amino acid substitution. Therefore, serine 76 is a crucial amino acid for proper regulation of the yeast enzyme. We constructed additional feedback-resistant enzyme forms of the yeast anthranilate synthase by site-directed mutagenesis of the conserved LLES sequence in the TRP2 gene. From analysis of these variants, we propose an extended sequence, LLESX10S, as the regulatory element in tryptophan-responsive anthranilate synthases from prokaryotic and eukaryotic organisms.