Clostridium perfringens type A isolates producing enterotoxin (CPE) are an important cause of food poisoning and non-food-borne human gastrointestinal (GI) diseases, including antibiotic-associated diarrhea (AAD).Recent studies suggest that C. perfringens type A food poisoning is caused by C. perfringens isolates carrying a chromosomal cpe gene, while CPE-associated non-food-borne GI diseases, such as AAD, are caused by plasmid cpe isolates. Those putative relationships, obtained predominantly with European isolates, were tested in the current study by examining 34 cpe-positive, C. perfringens fecal isolates from North American cases of food poisoning or AAD. These North American disease isolates were all classified as type A using a multiplex PCR assay. Furthermore, restriction fragment length polymorphism and pulsed-field gel electrophoresis genotyping analyses showed the North American AAD isolates included in this collection all have a plasmid cpe gene, but the North American food poisoning isolates all carry a chromosomal cpe gene. Western blotting demonstrated CPE expression by nearly all of these disease isolates, confirming their virulence potential. These findings with North American isolates provide important new evidence that, regardless of geographic origin or date of isolation, plasmid cpe isolates cause most CPE-associated AAD cases and chromosomal cpe isolates cause most C. perfringens type A food poisoning cases. These findings hold importance for the development of assays for distinguishing cases of CPE-associated food-borne and non-food-borne human GI illnesses and also identify potential epidemiologic tools for determining the reservoirs for these illnesses.Clostridium perfringens is a gram-positive, spore-forming, anaerobic bacterium that produces at least 15 different protein toxins (13,14,20,21,22). However, each individual C. perfringens isolate expresses only a defined subset of this total toxin repertoire, providing the basis for a commonly used classification scheme that assigns C. perfringens isolates to one of five types (A through E), based upon their ability to produce alpha-, beta-, epsilon-and iota-toxin (20,21).About 2 to 5% of all C. perfringens isolates, mostly belonging to type A, produce C. perfringens enterotoxin (CPE), a 35-kDa single polypeptide (12,17,26). These CPE-producing C. perfringens type A isolates are an important cause of enteric disease in both humans and domestic animals (18,19,25). Traditionally, these bacteria are most recognized as the cause of C. perfringens type A food poisoning, which currently ranks as the third most commonly identified food-borne disease in the United States (18). Considerable epidemiologic evidence implicates CPE as the virulence factor responsible for most (if not all) diarrheal and cramping symptoms associated with C. perfringens type A food poisoning (18). Furthermore, recent studies fulfilling the molecular Koch's postulates have provided unambiguous proof that CPE expression is required for the gastrointestinal (GI) virulence of ...