BACKGROUND & AIMS: Prominin-1(Prom1)/CD133 is used, alone or in combination with other cell surface markers, to identify and isolate stem cells from various adult tissues. We recently identified leucine-rich-repeat-containing G-protein-coupled receptor 5 (Lgr5) as a marker of the intestinal stem cells from which all cellular lineages of the gastrointestinal epithelium are derived. To determine whether there is a relationship between these markers, we investigated the intestinal expression pattern of Prom1/CD133 and created knock-in mice to visualize and trace Prom1(+) cells. METHODS: We analyzed Prom1 mRNA and protein expression among stem cells within intestinal crypts. Prom1/CD133 knock-in mice (Prom1(-mCherry-IRES-CreERT2) KI) were generated that express a fusion of red fluorescent protein mCherry with the C-terminus of Prom1. The knock-in allele also contains the tamoxifen-inducible CreERT2 recombinase, allowing for genetic tracing of progeny derived from Prom1-positive cells. RESULTS: In the small intestine, Prom1 mRNA was detected throughout the lower half of crypts and was not restricted to the rare stem cells that are sandwiched between Paneth cells. Prom1 protein was detected at the apical membranes of Lgr5(+) intestinal stem cells, but also on the transit-amplifying progenitors located above the Paneth cells. Analyses of the Prom1(-mCherry-IRES-CreERT2) KI mice showed that Prom1 is not exclusively expressed in Lgr5(+) intestinal stem cells but marks a much larger stem cell/transit-amplifying progenitor compartment. CONCLUSIONS: Prom-1 marks intestinal stem cells, as well as transit-amplifying progenitors, so it is not a specific marker for Lgr5(+) intestinal stem cells.