Primary adenocarcinoma of the urinary bladder is an uncommon neoplasm that can be indistinguishable morphologically from colorectal adenocarcinoma secondarily involving the bladder by direct extension or metastasis. In the current study, 17 enteric-type primary adenocarcinomas of the bladder were immunohistochemically examined for the expression of CDX2, villin and alpha-methylacyl coenzyme A racemase (AMACR), immunomarkers preferentially expressed in colorectal adenocarcinoma. For comparison, 17 secondary colorectal adenocarcinomas involving the bladder, 23 primary colorectal adenocarcinomas and 14 conventional urothelial carcinomas were similarly studied. The results show that all 40 (100%) colorectal adenocarcinomas expressed CDX2 and 39 (98%) expressed villin. The expression of these two immunomarkers was less frequent in primary bladder adenocarcinomas, observed in eight (47%) and 11 (65%) cases, respectively (P<0.0001 and P=0.0019, respectively). The frequency of positive AMACR immunostaining was similar between these two types of tumors, detected in 28 (70%) colorectal adenocarcinomas and 11 (65%) primary bladder adenocarcinomas (P=0.694). None of the urothelial carcinomas exhibited CDX2 or villin immunoreactivity; and only two (14%) showed positive staining for AMACR. These results demonstrate that CDX2 and villin are of diagnostic value in aiding in the distinction between primary adenocarcinoma of the bladder and secondary colorectal carcinoma. Lack of CDX2 and villin signals points strongly to a bladder primary.