Mitochondria were isolated from human liver biopsies obtained during surgical operations in patients with cholelithiasis or duodenal or gastric ulcer. Respiration with succinate, alpha-ketoglutarate or pyruvate + malate, respiratory control and P/O ratios were determined and compared with corresponding data for isolated rat liver mitochondria. It was found that in human liver mitochondria there was a faster oxygen uptake with succinate as substrate than in rat liver mitochondria. The oxygen uptake was somewhat less with alpha-ketoglutarate or pyruvate + malate. Respiratory control and P/O ratios were high for both preparations. Non-stimulated ATPase1 activity was low but increased in the basic pH range in accordance with earlier reports on rat liver mitochondria. Stimulation by physiologically occurring agents, viz. fatty acids and cholic acid and, with somewhat varying results, bilirubin, caused increases of ATPase activity at pH 7-8 while 2.4-dinitrophenol seemed to cause activation at other pH’s also. The stimulatory concentration ranges for the compounds tested were in agreement with those demonstrated earlier for rat liver mitochondria. In eleven patients with icterus of varying degree, caused by choledocholithiasis or carcinoma of the pancreas, respiration in liver mitochondria with alpha-ketoglutarate and pyruvate + malate was significantly diminished, while succinate respiration was maintained. Respiratory control was found to be lowered and ATPase activity increased. The results indicate that in cholestasis in the human, mitochondrial function is impaired in vivo in analogy with earlier findings in vitro, where bilirubin and other detergents have been added to rat liver mitochondria.