Non-growing cells can function as whole-cell biocatalysts for hydrogen (H-2) production, a process that has recently drawn much attention. In order to evaluate their potential as whole-cell biocatalysts, we compared the H-2-production capability of four Enterobacteriaceae strains (Citrobacter amalonaticus Y19, Escherichia coli K-12 MG1655, Escherichia coli DJT135, and Enterobacter aerogenes) under growing and non-growing conditions. We evaluated their H-2-production activity at varying temperatures (25-45 degrees C) and pH conditions (6.0-8.0) using glucose or formate as the carbon source. Under growing conditions with 10 mM glucose as a substrate, E. aerogenes exhibited the highest H-2-production activity (17.0 +/- 0.2 mu mol H-2 mg cell (1) h(-1)) among the four strains, but the final H-2 yield was similar (1.7-1.8 mol H-2 mol(-1) glucose) in all four strains. H-2 production in the four strains proceeded through a formate-dependent pathway that involved the formate hydrogen lyase (FHL) complex. Under non-growing conditions with 20 mM formate as a substrate, we obtained high H-2-production activities, in the range of 95.5-195.2 mu mol H-2 mg cell(-1) h (1), with E. coli DJT135 exhibiting the highest activity (195.2 mu mol H-2 mg(-1) h(-1)) at pH 6.0 and 45 degrees C. In contrast, using glucose as the carbon substrate in non-growing cell experiments greatly reduced the H-2-production activity to 6.1-7.7 mu mol H-2 mg cell (1) h(-1). This study indicated that formate is a better substrate than glucose for H-2 production by lion-growing cells, and that the H2-production performance among the strains did not vary significantly, with the exception of E. coli K-12 MG1655. (C) 2008 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights reserved.