The pattern of human retinoblastoma (RB1) gene protein expression was directly examined in normal and malignant human colorectal tissues and in seven colorectal carcinoma cell lines by immunohistochemistry using the mouse monoclonal antibody (RB-MAb-1) directed against the retinoblastoma protein (RB). This is the first demonstration of RB immunostaining in adult human colonic epithelium and colorectal carcinomas. Specificity using RB-MAb-1 was confirmed by western blot analysis, which showed bands of 110-116 kDa corresponding to the sizes of unphosphorylated and phosphorylated RB. RB staining of normal adult colonic epithelium was confined to the nucleus and was most intense in the transitional zone of the crypt, whereas lumenal cells (fully differentiated) were RB negative. Primary colorectal carcinomas and all the colon cancer cell lines stained positively for nuclear RB, but the expression was heterogeneous with varying fractions of RB negative cells present. Because we and others have previously shown that loss or inactivation of the RB1 gene is infrequent in colorectal carcinomas, reduced RB expression in such cells is probably due to a cellular regulatory mechanism. For example, RB negative cells may be those in early-G1 phase (known to have reduced RB levels) or growth-arrested cells that have differentiated. The ability to directly detect RB in primary colorectal carcinomas will permit assessment of whether heterogeneous expression of the RB1 gene product has prognostic significance for survival of patients with this cancer.