A protocol for detection of low numbers of E. coli O157:H7 in ground beef by nested PCR incorporating immunomagnetic separation (IMS) was developed. The protocol enabled detection of 24 colony-forming-units (CFU) in 10 g of seeded ground beef without enrichment cultivation. Differential centrifugation was used for maximally recovering the target CFU. Partial digestion of the resulting cell pellet with proteinase K at 37°C was used for the removal of beef tissue, which was required for the proper function of IMS. Within the range of 24 to 2400 CFU/10 g, a log linear relationship between the numbers of inoculated CFU and the integrated intensity of the nested PCR products was obtained with both shiga-like toxin (SLT) 1 and 2 primer pairs.