Due to the renewed interest in finding sustainable fuels or energy carriers, biohydrogen (Bio-H(2)) from biomass is a promising alternative. Fermentative Bio-H(2) production was studied in a continuous stirred tank reactor (CSTR) operated during 65.6 d with cheese whey (CW) as substrate. Three hydraulic retention times (HRTs) were tested (10, 6 and 4 h) and the highest volumetric hydrogen production rate (VHPR) was attained with HRT of 6 h. Therefore, four organic loading rates (OLRs) at a fixed HRT of 6 h were tested thereafter, being: 92.4, 115.5, 138.6 and 184.4 g lactose/L/d. The highest VHPR (46.61 mmol H(2)/L/h) and hydrogen molar yield (HMY) of 2.8 mol H(2)/mol lactose were found at an OLR of 138.6 g lactose/L/d; a sharp fall in VHPR occurred at an OLR of 184.4 g lactose/L/d. Butyric, propionic and acetic acids were the main soluble metabolites found, with butyric-to-acetic ratios ranging from 1.0 to 2.4. Bacterial community was identified by partial sequence analysis of the 16S rRNA and polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE). The results showed that at HRT of 10 h and 6 h were dominated by the Clostridium genus. The VHPR attained in this study is the highest reported value for a CSTR system using CW as substrate with anaerobic sludge as inoculum and represents a 33-fold increase compared to a previous study. Thus, it was demonstrated that continuous fermentative Bio-H(2) production from CW can be significantly enhanced by an appropriate selection of parameters such as HRT and OLR. Enhancements in VHPR are significant because it is a critical parameter to determine the full-scale practical application of fermentation technologies that will be used for sustainable and clean energy generation. (C) 2009 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights reserved.