l-Serine is usually produced from glycine. We have genetically engineered Escherichia coli to produce l-serine from glucose intracellularly. d-3-Phosphoglycerate dehydrogenase (PGDH, EC 1.1.1.95) in E. coli catalyzes the first committed step in l-serine formation but is inhibited by l-serine. To overcome this feedback inhibition, both the His(344) and Asn(346) residues of PGDH were converted to alanine and the mutated PGDH (PGDH(dr)) became insensitive to l-serine. However, overexpression of PGDH(dr) gave no significant increase of l-serine accumulation but, when l-serine deaminase genes (sdaA, sdaB and tdcG) were deleted, serine accumulated: (1) deletion of sdaA gave up to 0.03 mmol l-serine/g; (2) deletion of both sdaA and sdaB accumulated l-serine up to 0.09 mmol/g; and (3) deletion of sdaA, sdaB and tdcG gave up to 0.13 mmol l-serine/g cell dry wt.