Different concentrations of corn steep liquor (CSL) were tested in the cultivation of Zymomonas mobilis. Cell growth, ethanol production, and the formation of glucose–fructose oxidoreductase (GFOR) and glucono-δ-lactonase (GL), the enzymes responsible for the bio-production of gluconic acid and sorbitol, were examined. The cell yields using 25 g CSL l–1 and 40 g CSL l–1 (Y
X/S≈0.031 g g–1) were close to that obtained with 5 g yeast extract (YE) l–1. With 5 g CSL l–1 and 15 g CSL l–1, the nutritional limitation led to smaller Y
X/S. Using 100 g CSL l–1 produced an inhibitory effect on cell growth. Similar ethanol yields (92–95%) were calculated for each concentration of CSL and also for YE medium. The highest specific GFOR/GL activities (13.2–13.5 U g–1 dry cell) were reached with 25 g CSL l–1 and 40 g CSL l–1, values comparable to that achieved with 5 g YE l–1. The results confirm that CSL is an effective and cheap supplement for Z. mobilis medium, increasing the economic potential of a large-scale bio-production of sorbitol and gluconic acid by untreated Z. mobilis cells. The economic feasibility of the process is discussed.