No studies have investigated the mitochondrial function in permeabilized muscle fiber from cats. The aim of this study was to investigate tissue-specific and substrate-specific characteristics of mitochondrial oxidative phosphorylation (OXPHOS) capacity in feline permeabilized oxidative muscle fibers. Biopsies of left ventricular cardiac muscle and soleus muscle, a type I-rich oxidative skeletal muscle, were obtained from 15 healthy domestic cats. Enzymatic activity of citrate synthase (CS), a biomarker of mitochondrial content, was measured. Mitochondrial OXPHOS capacity with various kinds of non-fatty-acid substrates and fatty-acid substrate in permeabilized muscle fiber was measured by using high-resolution respirometry. CS activity in the heart was 3 times higher than in the soleus muscle. Mitochondrial state 3 respiration, ADP-stimulated respiration, with complex I-linked and complex I+II-linked substrates, respectively, was significantly higher in the heart than in the soleus muscle when normalized for muscle mass, but not for CS activity, indicating that greater capacity for mitochondrial OXPHOS with these non-fatty-acid substrates in the heart may depend on higher mitochondrial content. In contrast, the soleus muscle had higher mitochondrial state 3 respiration with fatty acids than the heart when normalized for CS activity, indicating greater capacity for fatty-acid oxidation per mitochondrion in the soleus. Our findings suggest that there are tissue- specific and substrate-specific quantitative and qualitative differences in mitochondrial OXPHOS capacity between the different types of oxidative muscles from cats.