A previously cloned gene of Saccharomyces cerevisiae, which complements the growth defect of a phosphoglucomutase (pgm1Δ/pgm2Δ) double deletion mutant on a pure galactose medium [Boles, E., Liebetrau, W., Hofmann, M. & Zimmermann, F. K. (1994) Eur. J. Biochem. 220, 83–96], was identified as the structural gene encoding N-acetylglucosamine-phosphate mutase. The complete nucleotide sequence of the gene, AGM1, and surrounding regions was determined. AGM1 codes for a predicted 62-kDa protein with 557 amino acids and is located on chromosome V adjacent to the known gene PRB1 encoding protease B. No extended nucleotide or amino acid sequence similarities could be found in the databases, except for a small region of amino acids with high similarity to the active-site consensus sequence of hexosephosphate mutases. Three putative pheromone-responsive elements have been identified in the upstream region of the AGM1 gene. The gene is essential for cell viability. An agm1 deletion mutant progresses through only approximately five cell cycles to form a ‘string' of undivided cells with an abnormal cell morphology resembling glucosamine auxotrophic mutants. Expression of the AGM1 gene on a multi-copy plasmid led to a significantly increased N-acetylglucosamine-phosphate mutase activity. Unlike over-expression of the AGM1 gene in a pgm1/pgm2 double deletion mutant which could restore phosphoglucomutase activity, over-expression of the PGM2 gene encoding the major isoenzyme of phosphoglucomutase did not increase N-acetylglucosamine-phosphate-mutase activity and did not restore growth of agm1 deletion mutant cells. Our observations indicate that the different hexosephosphate mutases of S. cerevisiae have partially overlapping substrate specifities but, nevertheless, distinct physiological functions.