PCR was used to establish a specific detection system for the non-pathogenic Escherichia coli strain Nissle 1917 (DSM6601), which is used as a probiotic drug against intestinal disorders and diseases. Five PCR assays have been developed which are based on the chromosomally encoded major fimbrial subunit genes fimA (type 1 fimbriae) and focA (F1C fimbriae), and the two small cryptic plasmids pMUT1 and pMUT2. The assays were validated by testing a collection of 354 different pathogenic and non-pathogenic E. coli strains from various origins, including E. coli K-12, fecal and environmental as well as pathogenic extraintestinal and intestinal E. coli strains. The most specific results were obtained with primers based on DNA sequences from plasmid pMUT2. The plasmid-based PCR assays described can be used to detect E. coli strain Nissle 1917 in feces from patients without prior cultivation.