This study presents high-resolution in situ cryo-electron microscopy (cryo-EM) images of porcine mitochondrial respiratory supercomplexes (SCs), which are crucial for ATP production through oxidative phosphorylation. The approach preserves the native mitochondrial environment, allowing for the determination of SC structures in their physiological states. Four main SC organizations—I,III,IV, I,III,IV, I,III,IV, and I,III,IV—are identified, with the latter two being new forms not observed in vitro. These SCs are primarily composed of 'protein–lipid–protein' interactions, which influence the local geometry of the surrounding membranes. The study captures multiple reactive intermediates within these SCs, providing insights into the Q-cycle mechanism in complex III and ubiquinone/ubiquinol exchange in complex I. Additionally, the impact of different mitochondrial diseases and pharmacological treatments on SC structures is assessed, revealing conformational changes in complexes I and III under various conditions. The findings highlight the dynamic nature of these SCs and their potential roles in physiological and pathological conditions.This study presents high-resolution in situ cryo-electron microscopy (cryo-EM) images of porcine mitochondrial respiratory supercomplexes (SCs), which are crucial for ATP production through oxidative phosphorylation. The approach preserves the native mitochondrial environment, allowing for the determination of SC structures in their physiological states. Four main SC organizations—I,III,IV, I,III,IV, I,III,IV, and I,III,IV—are identified, with the latter two being new forms not observed in vitro. These SCs are primarily composed of 'protein–lipid–protein' interactions, which influence the local geometry of the surrounding membranes. The study captures multiple reactive intermediates within these SCs, providing insights into the Q-cycle mechanism in complex III and ubiquinone/ubiquinol exchange in complex I. Additionally, the impact of different mitochondrial diseases and pharmacological treatments on SC structures is assessed, revealing conformational changes in complexes I and III under various conditions. The findings highlight the dynamic nature of these SCs and their potential roles in physiological and pathological conditions.