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 (x40), 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