The recent development of tissue microarray technology has potentiated large-scale retrospective cohort studies using archival formalin-fixed, paraffin-embedded tissues. A major obstacle to broad acceptance of microarrays is that they reduce the amount of tissue analyzed from a whole tissue section to a disk, 0.6 mm in diameter, that may not be representative of the protein expression patterns of the entire tumor. In this study, we examine the number to disks required to adequately represent the expression of three common antigens in invasive breast carcinoma--estrogen receptor, progesterone receptor, and the Her2/neu oncogene--in 38 cases of invasive breast carcinoma. We compared the staining of 2 to 10 microarray disks and the whole tissue sections from which they were derived and determined that analysis of two disks is comparable to analysis of a whole tissue section in more than 95% of cases. To evaluate the potential for using archival tissue in such arrays, we created a breast cancer microarray of 8 to 11 cases from each decade beginning in 1932 to the present day and evaluated the antigenicity of these markers and others. This array demonstrates that many proteins retain their antigenicity for more than 60 years, thus validating their study on archival tissues. We conclude that the tissue microarray technique, with 2-fold redundancy, is a valuable and accurate method for analysis of protein expression in large archival cohorts.