Simultaneous demonstration of acetylcholinesterase (AChE) and horseradish peroxidase (HRP) has been achieved by staining frozen sections with a modified Koelle-Friedenwald thiocholine method for AChE followed directly by the Graham-Karnovsky procedure for HRP. By using sodium sulfite instead of sodium sulfide in the AChE procedure, the final reaction product appears as black AChE granules that contrast sharply with the yellowish-brown HRP granules.