The determination of various reaction constants yields the following assay for the photometric evaluation of acid beta-galactosidase (measurement of the azoindoxyl dye at 540 nm after extraction with dimethylformamide or -acetamide): 1.5 mM 5-Br-4-Cl-3-indolyl-beta-D-galactoside (1 mg dissolved in 0.05 ml dimethylformamide) and 0.01-0.015 ml hexazotized p-rosaniline/ml in 0.1 M citric acid-phosphate buffer, pH 4. By means of this procedure it becomes evident that the activity of the enzyme differs considerably in various rat organs; NaCl does not influence acid beta-galactosidase. -- Similar results were obtained with the indigogenic method; indigo can be dissolved and measured photometrically as the azoindoxyl dye. The enzyme is suppressed by high concentrations of hexazotized p-roaniline to 50%; low concentrations do not inhibit; the same is true for ferricyanide-ferrocyanide employed in the indigogenic media. -- The effect of glutar- and formaldehyde on acid beta-galactosidase cannot be investigated with the azoindoxyl reaction since the azoindoxyl dye partially withstands extraction from fixed blocks of tissue. On the basis of the biochemical findings the azoindoxyl technique can be recommended for the histochemical demonstration of acid beta-galactosidase: 7.5 mg (1.5 mM) 5-Br-4-Cl-3-indolyl-beta-D-galactoside (dissolved in 0.25 ml dimethylformamide) and 0.05-0.15 ml hexazonium-p-rosaniline in 10 ml 0.1 M citric acid-phosphate buffer, pH 4. After incubation the sections can be treated with osmium tetroxide followed by dehydration and mounting in resins or can be mounted without prior osmification of the azoindoxyl dye in glycerin jelly. The osmium chelate resists treatment with organic solvents; the stability of the chelate depends on the concentration of hexazotized p-rosaniline. After fixation in glutaraldehyde or in a mixture of form- and glutaraldehyde acid beta-galactosidase can be exactly localized in the lysosomes of many rat organs. In comparison with the indigogenic, the metal precipitation and the simultaneous azocoupling reactions for the in situ detection of acid beta-galactosidase the azoindoxyl procedure is superior if fixed material is used; it is equivalent or inferior in connection with membrane technique. The biochemical azoindoxyl assay represents a useful method for combined qualitative and quantitative studies of acid beta-galactosidase.