Because of their rich composition in carbohydrates, lignocellulosic residues represent an interesting source of biomass to produce biohydrogen by dark fermentation. Nevertheless, pretreatments should be applied to enhance the solubilization of holocelluloses and increase their further conversion into biohydrogen. The aim of this study was to investigate the effect of thermo-alkaline pretreatment alone and combined with enzymatic hydrolysis to enhance biohydrogen production from sunflower stalks. A low increase of hydrogen potentials from 2.3 +/- 0.9 to 4.4 +/- 2.6 and 20.6 +/- 5.6 mL of H-2 g(-1) of volatile solids (VS) was observed with raw sunflower stalks and after thermo-alkaline pretreatment at 55 degrees C, 24 h, and 4% NaOH and 170 degrees C, 1 h, and 4% NaOH, respectively. Enzymatic pretreatment alone showed an enhancement of the biohydrogen yields to 30.4 mL of H-2 g(-1) of initial VS, whereas it led to 49 and 59.5 mL of H-2 g(-1) of initial VS when combined with alkaline pretreatment at 55 and 170 degrees C, respectively. Interestingly, a diauxic effect was observed with sequential consumption of sugars by the mixed cultures during dark fermentation. Glucose was first consumed, and once glucose was completely exhausted, xylose was used by the microorganisms, mainly related to Clostridium species.