The assembly of complex I (CI) with complexes III (CIII) and IV (CIV) of the mitochondrial respiratory chain (MRC) to configure I–III- or I–III–IV-containing supercomplexes (SCs) regulates mitochondrial energy efficiency and reactive oxygen species (mROS) production. However, whether the occurrence of SCs impacts on CI specific activity remains unknown to our knowledge. To investigate this issue, here we determined CI activity in primary neurons and astrocytes, cultured under identical antioxidants-free medium, from two mouse strains (C57Bl/6 and CBA) and Wistar rat, i.e. three rodent species with or without the ability to assemble CIV into SCs. We found that CI activity was 6- or 1.8-fold higher in astrocytes than in neurons, respectively, from rat or CBA mouse, which can form I–III₂–IV SC; however, CI activity was similar in the cells from C57Bl/6 mouse, which does not form I–III₂–IV SC. Interestingly, CII–III activity, which was comparable in neurons and astrocytes from mice, was about 50% lower in astrocytes when compared with neurons from rat, a difference that was abolished by antioxidants- or serum-containing media. CIV and citrate synthase activities were similar under all conditions studied. Interestingly, in rat astrocytes, CI abundance in I–III₂–IV SC was negligible when compared with its abundance in I–III-containing SCs. Thus, CIV-containing SCs formation may determine CI specific activity in astrocytes, which is important to understand the mechanism for CI deficiency observed in Parkinson’s disease.