Until now ubisemiquinones associated with NADH:ubiquinone oxidoreductase (complex I) have been reported to occur in isolated enzyme and in tightly coupled submitochondrial particles. In this report it is shown that ubisemiquinones are always detectable during steady-state electron transfer from NADH to ubiquinone, independent of the type of inner-membrane preparation used. The EPR signal of the rotenone-sensitive ubisemiquinones could be detected not only in coupled MgATP submitochondrial particles, but also in routine preparations of uncoupled submitochondrial particles and in mitochondria. The ubisemiquinone formation in coupled preparations was completely insensitive to uncouplers. The maximal radical concentration during steady-state electron transfer from NADH to quinone was equal to that of iron-sulphur cluster 2. Experiments with antimycin, myxothiazol and 2-thenoyltrifluoroacetone demonstrated that about half of this radical was associated with complex I, giving a ubisemiquinone concentration of about 0.5 mol semiquinone/mol cluster 2. Uncoupled submitochondrial particles, prepared by extensive sonification, never showed radical signals within 100 ms after mixing with NADH. This was due to the reversible inactivation of the enzyme, caused by elevated temperatures during sonification. In preparations with deliberately heat-inactivated complex I, no radical signals were detected within 200 ms after mixing with NADH; at 1 s, however, radical formation was maximal. Yet, depending on the procedure of reactivation of the complex, in preparations previously treated to inactivate them ubisemiquinone concentrations were always less than in untreated particles. When complex I was in the active state the ubisemiquinone signal was maximal within 40 ms. The results described in this report lead to the conclusion that ubisemiquinones form obligatory intermediates in the reaction of NADH dehydrogenase with ubiquinone.