Abstract Carcinogenesis is a multistep process characterized by the accumulation of genetic alterations that drive the transformation of normal cells to malignant cells. Telomeres are specialized nucleoprotein structures that function to protect and stabilize the ends of eukaryotic chromosomes by preventing chromosome fusions, masking double strand DNA break damage signals and preventing exonucleolytic chromosomal degradation. It is well established that telomere shortening is present in precursor and invasive stages of many carcinomas. Additionally, numerous investigations have demonstrated that altered telomere length predisposes cells to possess the properties necessary for metastasis formation, and therefore represents a predictor of clinical outcome. However, most of these studies have used assays that measure the average telomere length of all the cells in a particular population. To circumvent this problem, Baird et al. (Nature Genetics, 2003) previously described a PCR-based single telomere length analysis (STELA) method that could accurately measure the full spectrum of telomere lengths from a specific chromosomal arm. Although this method represents a substantial improvement over existing methods, the assay requires the use of radioactive material. Here, we describe a modification of the STELA assay which uses chemiluminescence instead of radioisotopes for product visualization, while maintaining the overall sensitivity of the detection of a wide range of telomere lengths. This new methodology has tremendous potential to illuminate the mechanisms that support the initiation of cancer and may provide an avenue for investigators to address the full extent and significance of telomere shortening in the tumorigenic process at the single chromosome level. The modified assay described here will allow for more widespread use of this technique among cancer researchers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3212.