Anaerobic fermentation for hydrogen (H(2)) production was studied in a two-stage fermentation system fed with different ripened fruit feedstocks (apple, pear, and grape). Among the feedstocks, ripened apple was the most efficient substrate for cumulative H(2) production (4463.7 mL-H(2) L(-1)-culture) with a maximum H(2) yield (2.2 mol H(2) mol(-1) glucose) in the first stage at a hydraulic retention time (HRT) of 18 h. The additional cumulative biohydrogen (3337.4 mL-H(2) L(-1)-culture) was produced in the second stage with the reused residual substrate from the first stage. The major byproducts in this study were butyrate, acetate, and ethanol, and butyrate was dominant among them in all test runs. During the two-stage system, the energy efficiency (H(2) conversion) obtained from mixed ripened fruits (RF) increased from 4.6% (in the first stage) to 15.5% (in the second stage), which indicated the energy efficiency can be improved by combined hydrogen production process. The RF could be used as substrates for biohydrogen fermentation in a two-stage (dark/dark) fermentation system. (C) 2010 Elsevier Ltd. All rights reserved.