A new procedure for the analysis and detection of phosphate-activated glutaminase (EC 3.5.1.2) by native electrophoresis has been developed. The method is based on the in situ detection of glutaminase activity in two different systems of native polyacrylamide gradient gels, containing 3-(3-cholamidopropyl)-dimethyl-ammonio-1-propane sulfonate (CHAPS) or Triton X-100 as nondenaturant detergents. Crude Triton X-100 extracts of mitochondria were resolved by electrophoresis. The enzyme was specifically revealed by incubation of the gel with glutamine and coupling the oxidation of the glutamate formed to the reduction of a tetrazolium dye, in the presence of glutamate dehydrogenase trapped in a 1% agar solid overlay. Both Ehrlich ascitic cell and mouse kidney glutaminases were resolved by native electrophoresis and specifically detected with the activity staining. Moreover, the redox-cycling staining was tested in solution, showing linearity with the amount of glutamate or glutaminase activity present. The method described could be a useful tool for native polyacrylamide gel electrophoresis of membrane proteins.