A method was developed to assess human telomere lengths at the individual cell level in tissue sections from standard formalin-fixed paraffin-embedded tissues. We coupled this method with immunofluorescence to allow the simultaneous identification of specific cell types. Validation of this in situ quantification method showed excellent agreement with the commonly used telomere repeat fragment-Southern blot method. The assay requires very few cells (~10 to 15). Thus, small tissue samples, including clinical biopsies,can be easily accommodated. In addition, the cells under study need not be actively cycling and there is no requirement for tissue disaggregation or cell culture. This method provides a more accurate assessment of telomere lengths than Southern blotting because confounding contributions from undesired cell types within tissue samples are avoided. Using this technique, we were able to perform the first comparison of relative telomere lengths in matched tumor versus normal epithelial cells within archival human prostate tissues. (Am J Pathol 2002, 160:1259–1268)