Escherichia coli possesses two hydrogenases, Hyd-3 and Hyd-4. These, in conjunction with formate dehydrogenase H (Fdh-H), constitute distinct membrane-associated formate hydrogenlyases, FHL-1 and FHL-2, both catalyzing the decomposition of formate to H2 and CO2 during fermentative growth. FHL-1 is the major pathway at acidic pH whereas FHL-2 is proposed for slightly alkaline pH. In this study, regulation of activity of these pathways by formate has been investigated. In cells grown under fermentative conditions on glucose in the presence of 30 mM formate at pH 7.5, intracellular pH was decreased to 7.1, the activity of Fdh-H raised 3.5-fold, and the production of H2 became mostly Hyd-3 dependent. These results suggest that at alkaline pH formate increases an activity of Fdh-H and of Hyd-3 both but not of Hyd-4.