Olive oil extraction produces a dark-colored wastewater that contains nutrients that can be further processed using biotechnology, in parallel with treatment for disposal. For instance, olive mill wastewater (OMW) can be used as a substrate for photofermentative hydrogen production by purple bacteria. A comparative study was investigated with several OMW samples front different olive oil mills in Western-Anatolia, Turkey. The composition of OMW varies significantly for each mill; thus, a detailed physicochemical analysis of each sample has been carried out. Subsequently, samples were assessed for their functioning in anaerobic photofermentative hydrogen production by Rhodobacter sphaeroides O.U.001. The highest hydrogen production potential (19.9 m(3) m(-3)) was obtained by the OMW sample with the highest organic content (mainly acetic acid, 9.71 kg m(-3)) and the highest carbon-to-nitrogen (C/N) molar ratio (73.8 M M-1). The organic content was found to be composed of primarily acetic, aspartic, and glutamic acids. There was a linear relationship between C/N ratio and hydrogen production potential across the different OMW samples. This study is unique due to the wide range of analyses of OMW samples and the comparison of many parameters for hydrogen production from wastewater. The results obtained throughout this study can aid in the design of systems using wastewater for biohydrogen production. Particularly, the C/N ratio was found to be the best parameter for choosing a proper substrate. (C) 2008 Elsevier Ltd. All rights reserved.