The tryptophan synthase-encoding gene, trpB, of Aspergillus nidulans was cloned and characterized. It was mapped to chromosome I, between the gene medA, which is required for sexual and asexual development, and an ORF encoding a protein with significant similarity to subunit B of vacuolar ATP synthases. The 5' untranslated region was found to be at least 142 nucleotides (nt) long, the poly(A) addition site was localized at position + 216 relative to the stop codon by sequencing of several independent cDNA clones. The trpB gene contains two exons separated by an intron of 105 nt, which is located close to the 5' end of the ORF. Directly upstream of the transcriptional start site, one well conserved potential binding site for the cross-pathway control transcriptional activator CPCA was found. The level of trpB transcript was shown to be regulated by cross-pathway control. A knockout mutant for trpB displays tryptophan auxotrophy, no trpB transcript is detectable, and development is perturbed to an extent that is dependent on the amount of tryptophan added to the medium. The trpB gene encodes a protein of 723 amino acids, with a calculated molecular weight of 77.6 kDa. The deduced amino acid sequence shows 72.6% similarity to the tryptophan synthase of Neurospora crassa. Most amino acid residues essential for catalytic activity in the tryptophan synthase of Salmonella typhimurium are conserved. The linker region joining the two domains of the enzyme is 13 residues longer than the longest connector found so far in tryptophan synthases from fungi.