The external pH appeared to be the main factor governing oxalic acid production by Aspergillus niger. A glucose-oxidase-negative mutant produced substantial amounts of oxalic acid as long as the pH of the culture was 3 or higher. When pH was decreased below 2, no oxalic acid was formed. The activity of oxaloacetate acetylhydrolase (OAH), the enzyme believed to be responsible for oxalate formation in A. niger, correlated with oxalate production. OAH was purified from A. niger and characterized. OAH cleaves oxaloacetate to oxalate and acetate, but A. niger never accumulated any acetate in the culture broth. Since an A. niger acuA mutant, which lacks acetyl-CoA synthase, did produce some acetate, wild-type A. niger is apparently able to catabolize acetate sufficiently fast to prevent its production. An A. niger mutant, prtF28, previously isolated in a screen for strains deficient in extracellular protease expression, was shown here to be oxalate non-producing. The prtF28 mutant lacked OAH, implying that OAH is the only enzyme involved in oxalate production in A. niger. In a traditional citric acid fermentation low pH and absence of Mn2+ are prerequisites. Remarkably, a strain lacking both glucose oxidase (goxC) and OAH (prtF) produced citric acid from sugar substrates in a regular synthetic medium at pH 5 and under these conditions production was completely insensitive to Mn2+.