To investigate the regional spread of microscopic tumor nodules in the mesorectum of patients with low rectal cancer, and to provide further pathological evidence for optimal procedure selection of radical resection for rectal cancer.Sixty-two patients with low rectal cancer underwent low anterior resection and total mesorectal excision (TME). Surgical specimens were sliced transversely on serial embedded blocks at 2.5-mm intervals, and stained with hematoxylin and eosin (HE). On whole-mount sections the mesorectum was divided into 3 regions: the outer region of the mesorectum (ORM), the middle region of the mesorectum (MRM), and the inner region of the mesorectum (IRM). Microscopic metastatic foci were investigated for metastatic mesorectal region, frequency, types, involvement of the lymphatic system, and correlation with the primary tumor. Tumor-suspect nodules previously considered disease free by HE stain on whole-mount section were examined by in situ hybridization (ISH) on tissue microarray (TMA) through detecting mRNAs of CEA and CK20 with non radioactive biotin-tagged oligonucleotide probes.Microscopic spread of the tumor was observed in 50.0 percent of patients (31 out of 62, 24 by HE stain on whole-mount section and 7 by ISH on TMA) and that in the ORM was observed in 38.7 percent of the patients (24 out of 62, 16 observed by HE stain on whole-mount section and 8 by ISH on TMA). Microscopic tumor foci spread in the circumferential resection margin (CRM) occurred in 8.1 percent of the patients (5 out of 62, 4 observed by HE stain on whole-mount section and one by ISH on TMA), and distal mesorectum (DMR) involvement was detected in 6.5 percent (4 out of 62, all observed by HE stain on whole-mount section), with the spread extending to within 3 cm from the lower margin of the tumor. Most (26 of 31) of the patients with microscopic spread in mesorectum had TNM Stage III diseases.The results of the present study support the theory that complete excision of the mesorectum without destruction of the ORM is essential for surgical management of low rectal cancer, and an optimal DMR clearance resection margin of no less than 4 cm was referenced. Five patients with microscopic tumor nodule spread in the CRM observed in the study suggested that microscopic metastases exist in pelvic lateral areas and in the mesorectum simultaneously, indicating the significance of preoperative and/or postoperative radiochemotherapy.