Abstract Background.—Immunohistochemical staining for tumor-associated proteins is widely used for the identification of novel prognostic markers. Recently, a tissue-conserving, high-throughput technique, tissue microarray, has been introduced. This technique uses 0.6-mm tissue core biopsy specimens, 500 to 1000 of which are brought into a new paraffin array block, which can be sectioned up to 100 times. Methods.—We evaluated the tissue microarray technique for immunohistochemical analysis in 20 rectal cancers. Immunohistochemical staining was performed for the proliferation marker Ki-67 and the tumor suppressor protein p53 in whole tissue sections and in tissue core biopsy specimens. Results.—The whole tissue sections were assessed by counting all cells in 10 high-power fields (×40), which resulted in a mean fraction of Ki-67–expressing tumor cells of 0.81 (range, 0.54–1.0). p53 expression assessed in whole tissue sections showed nuclear staining in 15 (75%) of 20 rectal carcinomas. For the tissue microarray technique, a median of 3 (range, 3–5) 0.6-mm tissue core biopsy specimens were studied from each of the 20 tumor specimens. The tissue microarray method gave a mean Ki-67 expression of 0.85 (range, 0.50–1.0) in tumor cell nuclei and showed p53 protein expression in the same 15 of 20 tumors as in the whole tissue sections. Conclusion.—We conclude that the tissue microarray technique for immunohistochemical staining in rectal cancer yields staining of good quality and expression data for Ki-67 and p53 comparable to those obtained with whole tissue staining. The feasibility of tissue microarray thus enables time- and tissue-preserving studies of multiple markers in large tumor series.