ABSTRACTA membrane filtration‐immunostaining technique was developed for detection and direct enumeration of enterotoxigenic Clostridium perfringens in beef. The procedure combined a microcolony technique with an indirect alkaline phosphatase conjugated antibody visualization system. Precooked ground beef samples containing enterotoxigenic C. perfringens cells were diluted in phosphate buffered saline containing 0.1% Tween 80 and filtered through a 0.45 μm cellulose nitrate filter. The membranes were placed onto the surface of solid Duncan and Strong medium and incubated anaerobically for 4–18 h at 37C. The membranes were sequentially overlaid with C. perfringens anti‐enterotoxin and an alkaline phosphatase conjugated antibody resulting in a chromogenic reaction that enabled detection and enumeration. The membrane immunostaining technique proved to be sensitive enough to detect as low as 2 CFU/g of enterotoxigenic C. perfr