Five bacterial isolates were tested for their ability to generate hydrogen during anaerobic fermentation with various carbon sources. One isolate from sheep rumen was identified as Escherichia coli and four isolates belonged to Clostridium spp. Glucose, arabinose, ribose, xylose, lactose and cellobiose were used as carbon sources. Results showed that all bacterial strains could utilize these compounds, although the utilization of pentoses diminished growth yield. The excretion of monocarboxylic acids (acetate, propionate, formiate, butyrate) into medium was changed after replacing glucose by other carbon sources. Di- and tricarboxylic acids were excreted in negligible amounts only. Spectra of excreted carboxylic acids were unique for each strain and all carbon sources. All isolates produced H2 between 4—9 mmol·L−1 during the stationary phase of growth with glucose as energy source. This value was dramatically reduced when pentoses were used as carbon source. Lactose and cellobiose, starch and cellulose were suitable substrates for the H2 production in some but not all isolates. No H2 was produced by proteinaceous substrate, such as blood. Results show that both substrate utilization and physiological responses (growth, excretion of carboxylates, H2 production) are unique functions of each isolate.