Previously, we investigated ubisemiquinone (SQ) EPR spectra associated with NADH‐ubiquinone oxidoreductase (complex I) in the tightly coupled bovine heart submitochondrial particles (SMP). Based upon their widely differing spin relaxation rate, we distinguished SQ spectra arising from three distinct SQ species, namely SQNf (fast), SQNs (slow), and SQNx (very slow). The SQNf signal was observed only in the presence of the proton electrochemical gradient , while SQNs and SQNx species did not require the presence of . We have now succeeded in characterizing the redox and EPR properties of SQ species in the isolated bovine heart complex I. The potentiometric redox titration of the g z,y,x = 2.00 semiquinone signal gave the redox midpoint potential (E m) at pH 7.8 for the first electron transfer step [E m1(Q/SQ)] of −45 mV and the second step [E m2(SQ/QH2)] of −63 mV. It can also be expressed as [E m(Q/QH2)] of −54 mV for the overall two electron transfer with a stability constant (K stab) of the SQ form as 2.0. These characteristics revealed the existence of a thermodynamically stable intermediate redox state, which allows this protein‐associated quinone to function as a converter between n = 1 and n = 2 electron transfer steps. The EPR spectrum of the SQ species in complex I exhibits a Gaussian‐type spectrum with the peak‐to‐peak line width of ∼6.1 G at the sample temperature of 173 K. This indicates that the SQ species is in an anionic Q ▪− state in the physiological pH range. The spin relaxation rate of the SQ species in isolated complex I is much slower than the SQ counterparts in the complex I in situ in SMP. We tentatively assigned slow relaxing anionic SQ species as SQNs, based on the monophasic power saturation profile and several fold increase of its spin relaxation rate in the presence of reduced cluster N2. The current study also suggests that the very slowly relaxing SQNx species may not be an intrinsic complex I component. The functional role of SQNs is further discussed in connection with the SQNf species defined in SMP in situ.