1. Assay conditions are described for the ATP-dependent, uncoupler-sensitive, energy-linked reduction of NAD(+) by succinate, dl-alpha-glycerophosphate or d-lactate in membranes from aerobically grown Escherichia coli. 2. The reaction may be demonstrated in electron-transport particles (ET particles) from cells grown in glycerol, but not in depleted particles washed in low-ionic-strength buffer, or in ET particles from cells grown in glucose. 3. The latter two classes of particles have low specific activities of ATPase (adenosine triphosphatase), succinate dehydrogenase, dl-alpha-glycerophosphate dehydrogenase and d-lactate dehydrogenase relative to undepleted ET particles from cells grown in glycerol. 4. Reconstitution of energy-linked NAD(+) reduction in particles from cells grown in glucose was done by: (a) addition of the high-speed supernatant fraction from sonicates of the same cells; (b) addition of a protein fraction, precipitated by (NH(4))(2)SO(4) from this supernatant, or (c) addition of an (NH(4))(2)SO(4)-precipitated fraction from the low-ionic-strength wash of particles from cells grown in glycerol. 5. The use of (NH(4))(2)SO(4)-precipitated fractions from ATPase- or succinate dehydrogenase-deficient mutants grown in glycerol in the above reconstitution indicated that failure to demonstrate the reaction in particles from cells grown in glucose was a result of inadequate activities of appropriate dehydrogenases, rather than of ATPase. 6. Energy-linked NAD(+) reduction could be demonstrated in particles from a ubiquinone-deficient mutant only after restoration of NADH oxidase activity by adding ubiquinone-1. 7. The measured rate of the energy-linked reaction in particles from a haem-deficient mutant, however, was not stimulated after the ATP- and haematin-dependent acquisition of functional cytochromes. 8. Results are interpreted as evidence of the ubiquinone-dependent, but cytochrome-independent, nature of the site I region of the respiratory chain in E. coli.