For a further understanding of the inhibitory effect of NaCl on hog kidney mitochondrial monoamine oxidase (MAO), the activity for benzylamine as substrate was assayed spectrophotometrically in the absence and presence of NaCl for mitochondrial outer membrane preparations as well as whole mitochondria. The effect of CaCl2 was also examined for comparison. The inhibition by NaCl but not CaCl2 was strongly pH dependent. The pH dependence of the inhibitory effect of NaCl in phosphate buffer was parallel to the pH dependence of the MAO activity itself. The point at which the slope of the Arrhenius plot in the absence of NaCl decreases with increasing temperature was to be 32.3 degrees C at pH 7.0 and 30.4 degrees C at pH 7.5 in phosphate buffer, while the Arrhenius plot in the presence of NaCl exhibited discontinuities without change in the slope in small temperature ranges, 39.2 degrees C-40.0 degrees C and 33.0 degrees C-34.2 degrees C. It was estimated that the inhibitory effect of NaCl was due to a pH and temperature sensitive cooperative state change involving MAO protein and boundary lipids, while the effect of CaCl2 could be induced by specific Ca2+ binding to acidic phospholipids.