A 5' nuclease assay was developed to detect Lawsonia intracellularis in porcine fecal samples. The specific probe and primers were chosen by using the 16S ribosomal DNA gene as a target. The 5' nuclease assay was used with a total of 204 clinical samples, and the results were compared to those of immunohistochemistry (IM) on ileal sections of the same animals. There was 91% agreement between the results of IM and the 5' nuclease assay. In the 5' nuclease assay, 111 (54%) of the pigs tested positive for L. intracellularis infection, with a mean cycle threshold (Ct) value of 27.2, whereas 98 (48%) of the pigs tested positive by IM. On average, the Ct and DeltaRn values for the positive samples were 27.2 (standard deviation [SD], 3.7) and 1.6 (SD, 0.7), respectively. A Ct value of 27.2 corresponds to a fecal excretion of approximately 10(7) L. intracellularis cells per g of feces. Furthermore, a total of 40 fecal samples derived from a herd known to be free from infection with L. intracellularis all tested negative, with a Ct value of 40. By using a Ct value of 36 as the cutoff limit, the detection limit of the assay was 1 L. intracellularis cell per PCR tube. In conclusion, the 5' nuclease assay that has been developed represents an applicable fast method for detection of L. intracellularis in fecal samples, with a sensitivity and specificity comparable to those of IM.