In the lysis of formate by the immobilized formate hydrogen lyase (FHL) of Escherichia coli, generation of hydrogen was dependent on the addition of a small amount of glucose, tetrathionate, thiosulfate, or any one of the dicarboxylic acids such as fumarate, malate, succinate, and oxalocetate. During lysis, activities of anerobic reductases of E. coli, i.e., fumarate --> succinate and tetrathionate --> thiosulfate were evident as cells converted all acids and glucose into succinate and tetrathionate into thiosulfate. However, the cells also reduced fumarate and tetrathionate and converted glucose into succinate in the absence of formate. The presence of endogenous reducing substrate in the cell was also indicated by the spontaneous reduction of methylene blue (MB) by the cells which was stimulated by formate. MB reduction was not dependent of FHL activity and the presence of succinate of thiosulfate. It appeared that electron flow by endogenous substrate oxidation could reduce oxidants of high (MB) and low (fumarate and tetrathionate) redox potentials. This pathway is overlapping with the electron flow pathway of FHL probably due to the presence of common or similar electron transport carriers of close redox potentials. Thus, hydrogen production was terminated due to channeling of electrons from formate to the oxidants produced by the endogenous substrate but not to hydrogenase. The presence of excess thiosulfate and succinate prevented the channeling and maintained the flow of electrons for the reduction of hydrogen ion.