The cyclin-dependent kinase inhibitor p27^Kip1 can inhibit the G₁ to S transition of the cell cycle and is a putative tumor suppressor. However, our laboratory found that a variety of human cancer cell lines express relatively high levels of this protein and that this is often associated with increased expression of cyclin D1 or cyclin E. Therefore, in the present study we analyzed by immunohistochemistry the expression of p27^Kip1 in a series of human tissue samples representing various stages of colon carcinogenesis, using 20 samples of normal colon mucosa, 20 hyperplastic polyps, 19 samples of adenomatous polyps, and 40 samples of various types of colorectal carcinomas. Parallel immunostaining was done for cyclin D1 and also for Ki67 to evaluate cell proliferation. An additional 17 human colon carcinoma samples, together with paired adjacent normal mucosa samples, were analyzed for levels of expression of the p27^Kip1 protein by Western blot analysis, and 7 of these pairs of samples were examined by Northern blot analysis for levels of p27^Kip1 mRNA. We did not find a positive or negative correlation between p27^Kip1 expression and cell proliferation in the normal mucosa and tumor samples. There was, however, an inverse correlation between p27^Kip1 and Ki67 expression in the lymphoid follicles present in the colonic mucosa. There was no evidence for a consistent increase or decrease in p27^Kip1 expression in the mucosal cells during colon carcinogenesis, because the mean values for percentage p27^Kip1-positive cells were similar in the normal mucosa, adenomatous polyps, and carcinoma samples. This is in contrast to Ki67 and cyclin D1 expression, which did show significant increases in mean values with tumor development. A subset (35%) of the carcinomas displayed diffuse cytoplasmic staining, in addition to nuclear staining, for p27^Kip1, and in these cases the percentage of cells that were positive for p27^Kip1 was higher than in cases that had only nuclear staining. There was a significant correlation between p27^Kip1 expression and tumor grade; i.e., well and moderately differentiated carcinomas had high p27^Kip1 expression, whereas poorly differentiated carcinomas had lower expression. The Western blot analysis data on p27^Kip1 expression confirmed this correlation. Comparisons of Northern and Western blots did not show a correlation between the level of p27^Kip1 mRNA and the corresponding protein, a finding consistent with evidence that the p27^Kip1 protein is regulated mainly via a posttranscriptional mechanism. The immunostaining studies revealed a significant correlation between high p27^Kip1 protein expression and high cyclin D1 expression in the adenomatous polyps and in the subset of carcinomas that had only nuclear p27^Kip1 expression. This may reflect the existence of a homeostatic feedback mechanism that is lost in the high-grade carcinomas that express low levels of p27^Kip1.