Escherichia coli grown anaerobically for osmotic studies upon increased osmolarity in alkaline medium carried out H+-K+-exchange in two steps, the first of which was DCCD1 sensitive and osmo-dependent and had the 2H(+)/K+ stoichiometry. H+-efflux in the presence of protonophore (CCCP) upon increase of osmolarity was shown to be high and inhibited by DCCD, whereas H+-efflux induced by a decrease of osmolarity was small and not inhibited by DCCD. The 2H(+)/K+-exchange was absent in trkA and uncA mutants. In uncB mutant 2H(+)/K+-exchange was not DCCD-and osmosensitive. Competition between DCCD and osmoshock on inhibition of 2H(+)/K+-exchange was found. Osmosensitivity of this exchange disappeared in spheroplasts. Osmosensitivity of both 2H(+)/K+-exchange and the F0F1 and osmoregulation of the F0F1 via F-0 and a periplasmic space are postulated.