The uptake of homologous DNA by Haemophilus influenzae was studied as a function of the proton motive force in completely competent cultures in the pH range of 6 to 8. The composition and magnitude of the proton motive force were varied by using the ionophores valinomycin and nigericin (in the presence of various potassium ion concentrations) and by using protonophores. No interaction of the ionophores with the DNA transformation system itself was observed. Either component of the proton motive force, the electrical potential or the pH gradient, can drive the uptake of DNA, and the extent of the uptake of DNA is ultimately determined by the total proton motive force. The transformation frequency increases with the proton motive force, which reaches a maximum value at around -130 mV. These results are consistent with an electrogenic proton-DNA symport mechanism, but direct evidence for such a system is not available. The proton motive force was followed during competence development of H. influenzae at pH 8. In the initial phase (up to 50 min), the proton motive force remained constant at about -90 mV, whereas the transformation frequency rose steeply. In the second phase, the proton motive force increased. The transformation frequency in this phase increased with the proton motive force, as in completely competent cultures. These observations and the observed inhibition by NAD of both the proton motive force and the transformation frequency indicate that structural components of the competent state are formed in the initial phase of competence development, whereas the second phase is characterized by an increase of the proton motive force.