Physostigmine is a plant alkaloid of great interest as a therapeutic candidate for the treatment of Alzheimer’s disease. Fortunately, this compound is also produced by Streptomyces gl griseofuscus NRRL 5324 during submerged cultivation. A fermentation process that used chemically defined medium was therefore developed for its production. By means of statistical experimentation, the physostigmine titer was quickly increased from 20 mg/l to 520 mg/l with a culture growth of 19 gl dry cell weight on the shake-flask scale. Further medium optimization resulted in a yield of 790 mg/l in a 23-1 bioreactor using a batch process. A titer of 880 mg/l was attained during scale-up in a 800-1 fermenter by employing a nutrient-feeding strategy. This production represents a 44-fold increase over the yield from the initial process in shake-flasks. The defined-medium fermentation broth was very amenable to downstream processing.