This study investigated the determination of hydrogen production by three H-2-producing microorganisms (Thermotoga maritima, Thermococcus kodakarensis KOD1 and Enterobacter cloacae) cultured in 116-mL serum bottles. A gas pressure manometer was used to measure total pressure in the serum-bottle headspace. It was demonstrated that total pressure is the sum of the saturation pressure of water, the pressure expansion of gases, and the partial pressures of H-2 (PgH2) and CO2 (PgCO2). A linear relationship was established between the partial pressure of H-2 measured by gas chromatography and the sum of the partial pressures of H-2 and CO2 measured by the manometer. When pH of culture medium was not controlled (pH decreased from 7 to 5), the PgH2/PgCO2 ratio was close to stoichiometric H-2/CO2 yield ratio of the most plausible metabolic pathways of each strain. PgH2/PgCO2 values were 1.7, 1.9 and 0.9 for T. maritima, T. kodakarensis and E. cloacae, respectively. In these experimental conditions, PgH2 can be deduced from total pressure measured by manometer.