The kinetics of regulation mitochondrial respiration by external ADP in permeabilized hepatocytes was studied further. In digitonin-permeabilized hepatocytes, the apparent Km for ADP in regulation of respiration was decreased from 275 +/- 35 microM in control to 48 +/- 8 microM by a treatment with trypsin (15 min, 0.125 mg/ml). In liver tissue homogenates, trypsin treatment similarly decreased the Km value for ADP. These results show that ADP diffusion in hepatocytes may be retarded due to some unknown cytoplasmic trypsin-sensitive protein factor(s) which may be lost during isolation of mitochondria. Since we have previously reported a limited permeability of the outer mitochondrial membrane in isolated hepatocytes (Saks et al. 1995, Biochem. Biophys. Res. Commun., 208, 919-926), we conclude that an important site of control of respiration in liver cells in vivo is located at the porin channels of the outer mitochondrial membrane.