The production of riboflavin by Eremothecium ashbyii grown in a chemically defined medium in batch culture was affected by pH of the medium. Highest yields were obtained at constant pH of 4.5 and 5.5, while little or no riboflavin was detected at either pH 3.5 or 8.5. The medium pH also affected cell morphology. When the organism was grown in a stirred tank reactor and an airlift vessel at pH 4.5 very similar levels of riboflavin were obtained.