Purpose: The intestinal organism Oxalobacter formigenes is unique in using oxalate as its primary carbon and energy source. Intestinal colonization with O. formigenes may have clinical significance by decreasing intestinal oxalate and its absorption, thereby influencing the concentration of oxalate in plasma and urine, and the development of calcium oxalate stone disease. Because the oxalate content of the diet varies considerably, we hypothesized that the number of O. formigenes and amount of oxalate would vary in feces. Materials and Methods: To enumerate the number of O. formigenes in feces an accurate and reproducible real-time polymerase chain reaction assay was developed to quantify O. formigenes DNA. Stool samples were obtained from 10 colonized individuals to determine the levels of O. formigenes by this assay and the oxalate content by ion chromatography. Results: Concentrations of O. formigenes ranged from lower than the limit of detection of 5 × 103 to 1.04 × 109 cells per gm stool. The total oxalate content of stool samples varied from 0.1 to 1.8 mg/gm and fecal water oxalate varied from 60 to 600 μM. All parameters measured varied within each stool collection, among stool collections on different days and among individuals. Notably in 7 of 10 individuals at least 1 stool sample contained no detectable O. formigenes. In addition, 7 of 10 subjects had a fecal colonization of less than 4 × 104 per gm stool. Conclusions: This study demonstrates that there is intrastool and interstool sample variability in the amount of O. formigenes measured by real-time polymerase chain reaction that did not correlate with the quantity of oxalate in stool. Most subjects had a fecal colonization of less than 4 × 104 per gm stool. © 2007 American Urological Association.