Gluconic acid fermentation by Aspergillus niger has been investigated using untreated and treated Indian cane molasses. The yield of gluconic acid was found to be reduced using an untreated molasses medium compared to a defined medium. Hence, molasses was subjected to various pretreatment techniques. Pretreatment reduced the levels of various cations and anions. As the synthesis of gluconic acid has been observed to be influenced more by cations than anions, the effect of various metal ions, viz., copper, iron, zinc, manganese, calcium, and magnesium on the yield of gluconic acid has been critically examined in both untreated and treated cane molasses. These results have been compared with a defined medium. The yield of gluconic acid was influenced more by a combination of metal ions rather than individual ions. Potassium ferrocyanide treatment gave the most promising results compared to other treatment techniques. © 1994 Springer-Verlag.