B-cell leukemia 3 (Bcl-3) is a member of the inhibitor of κB family, which regulates a wide range of biological processes by functioning as a transcriptional activator or as a repressor of target genes. Elevated expression, sustained nuclear accumulation, and uncontrolled activation of Bcl-3 causes increased cellular proliferation or survival, dependent on the tissue and type of stimuli. We retrospectively reviewed patients who were diagnosed with colorectal cancer at Skåne University Hospital in Malmö between 1st of January 1990 and 31st of December 1991. Bcl-3 localization in colorectal cancer was assessed by immunohistochemistry on tissue microarray and freshly isolated colon from patients. Correlation between Bcl-3 localization and clinicopathological parameters of the cohort were evaluated using the Spearman rank-order correlation coefficient. In addition, Bcl-3 expression and localization in colon adenocarcinoma cells were analysed by western blot, immunohistochemistry and subcellular fractionation separately. We found that Bcl-3 was mainly localized in the cytoplasm in the tumour tissue isolated from colon cancer patients. Normal colon samples from the same patients showed Bcl-3 localization in the nucleus. In three out of six colon cancer cell lines, we detected elevated levels of Bcl-3. In these cell lines Bcl-3 was accumulated in the cytosol. We confirmed these findings by analysing Bcl-3 localization in a colon tissue micro array consisting of 270 cases. In these samples Bcl-3 localization correlated with the proliferation marker Ki-67, but not with the apoptotic marker Caspase 3. These findings indicate that analysis of the subcellular localization of Bcl-3 could be a potential-early diagnostic marker in colon cancer.