Single-read sequence analysis of the termini of eight randomly picked clones of Ashbya gossypii genomic DNA revealed seven sequences with homology to Saccharomyces cerevisiae genes (15% to 69% on the amino acid level). One of these sequences appeared to code for the carboxy-terminus of threonine synthase, the product of the S. cerevisiae THR4 gene (52.4% identity over 82 amino acids). We cloned and sequenced the complete putative AgTHR4 gene of A. gossypii. It comprises 512 codons, two less than the S. cerevisiae THR4 gene. Overall identity at the amino acid sequence level is 67.4%. A continuous stretch of 32 amino acids displaying complete identity between these two fungal threonine synthases presumably contains the pyridoxal phosphate attachment site. Disruption of the A. gossypii gene led to threonine auxotrophy, which could be complemented by transformation with replicating plasmids carrying the AgTHR4 gene and various S. cerevisiae ARS elements. Using these plasmids only very weak complementation of a S. cerevisiae thr4 mutation was observed. Investigation of sequences adjacent to the AgTHR4 gene identified three additional ORFs. Surprisingly, the order and orientation of these four ORFs is conserved in A. gossypii and S. cerevisiae.