We investigated the enzyme cytochemical localization of sarcosine oxidase (SOX) in the liver and kidney of several mammals using a cerium technique. First we measured the enzyme activities in the liver and kidney of several mammals and in several organs of mice. The highest activity was found in the Chinese hamster, followed by the mouse. Therefore, we used hamster and mouse tissues for enzyme cytochemistry. The liver and kidneys were fixed by perfusion with various concentrations of glutaraldehyde for 10 min. Tissue slices were incubated in reaction medium consisting of 50 mM TRIS-maleate buffer (pH 7.8), 9 mM sodium azide, 9.8 mM sarcosine, 25 microM FAD, 2 mM cerium chloride, 0.002% saponin, and 0.003% Triton X-100 for 0.5-8 h at 37 degrees C. Optimum staining reaction was obtained in tissues fixed with 0.2% glutaraldehyde, followed by incubation for 2-4 h. Electron-dense reaction products were present exclusively in peroxisomes. Within the peroxisomes strong reactions were observed in the matrix subjacent to the limiting membrane decreasing toward the center. The staining reaction was completely inhibited by 2 mM N-bromosuccinimide. These results indicated that SOX is a peroxisomal enzyme and that the enzyme might be associated with the peroxisomal membrane.