A rapid, reliable method for the simultaneous separation of adenosine deaminase, adenylate kinase, and carbonic anhydrase II by agarose gel electrophoresis is presented. This method uses a double origin sample application system. Unreduced sample extracts for adenylate kinase analysis are applied 13.0 cm from the anode. Reduced sample extracts for the remaining proteins of interest are applied 7.0 cm from the anode. The use of applicator foils and an increased voltage gradient result in superior resolution, linearity, and band sharpness of the allozyme patterns. Further, there is no masking of the adenylate kinase 2 band as a result of the use of a reducing agent, and carbonic anhydrase II is resolved without interference from hemoglobin as has been observed with other multisystem methods.