Background. Corn grain is an important renewable source for bioethanol production in the USA. Corn ethanol is currently produced by steam liquefaction of starch-rich grains followed by enzymatic saccharification and fermentation. Corn stover (the non-grain parts of the plant) is a potential feedstock to produce cellulosic ethanol in second-generation biorefineries. At present, corn grain is harvested by removing the grain from the living plant while leaving the stover behind on the field. Alternatively, whole corn plants can be harvested to cohydrolyze both starch and cellulose after a suitable thermochemical pretreatment to produce fermentable monomeric sugars. In this study, we used physiologically immature corn silage (CS) and matured whole corn plants (WCP) as feedstocks to produce ethanol using ammonia fiber expansion (AFEX) pretreatment followed by enzymatic hydrolysis (at low enzyme loadings) and cofermentation (for both glucose and xylose) using a cellulase-amylase-based cocktail and a recombinant Saccharomyces cerevisiae 424A (LNH-ST) strain, respectively. The effect on hydrolysis yields of AFEX pretreatment conditions and a starch/cellulose-degrading enzyme addition sequence for both substrates was also studied. Results. AFEX-pretreated starch-rich substrates (for example, corn grain, soluble starch) had a 1.5-3-fold higher enzymatic hydrolysis yield compared with the untreated substrates. Sequential addition of cellulases after hydrolysis of starch within WCP resulted in 15-20% higher hydrolysis yield compared with simultaneous addition of hydrolytic enzymes. AFEX-pretreated CS gave 70% glucan conversion after 72 h of hydrolysis for 6% glucan loading (at 8 mg total enzyme loading per gram glucan). Microbial inoculation of CS before ensilation yielded a 10-15% lower glucose hydrolysis yield for the pretreated substrate, due to loss in starch content. Ethanol fermentation of AFEX-treated (at 6% w/w glucan loading) CS hydrolyzate (resulting in 28 g/L ethanol at 93% metabolic yield) and WCP (resulting in 30 g/L ethanol at 89% metabolic yield) is reported in this work. Conclusions. The current results indicate the feasibility of co-utilization of whole plants (that is, starchy grains plus cellulosic residues) using an ammonia-based (AFEX) pretreatment to increase bioethanol yield and reduce overall production cost. © 2010 Shao et al; licensee BioMed Central Ltd.