Whey, an abundant byproduct of the dairy industry, contains large amounts of protein and lactose which could be used for fuel ethanol production. We have investigated a new organism as a candidate for such fermentations: recombinant Escherichia coli containing the genes encoding the ethanol pathway from Zymomonas mobilis. The highest level of ethanol achieved, 68 g/L, was produced after 108 hours in Luria broth containing 140 g lactose/L. Fermentations of lower lactose concentrations were completed more rapidly with approximately 88% of theoretical yields. Reconstituted sweet whey (60 g lactose/L) was fermented more slowly than lactose in Luria broth requiring 144 hours to produce 26 g ethanol/L. Supplementing sweet whey with a trace metal mix and ammonium sulfate reduced the required fermentation time to 72 hours and increased final ethanol concentration (28 g ethanol/L). By adding proteinases during fermentation, the requirement for ammonia was completely eliminated, and the rate of fermentation further improved (30 g ethanol/L after 48 hours). This latter increase in rate of ethanol production and ethanol yield are presumed to result from incorporation of amino acids released by hydrolysis of whey proteins. The fermentation of sweet whey by ethanologenic E. coli reduced the nonvolatile residue by approximately 70%. This should reduce biological oxygen demand and reduce the cost of waste treatment. Whey supplemented with trace metals and small amounts of proteinase may represent an economically attractive feedstock for the production of ethanol and other useful chemicals.