Using comparative proteomic analysis we have identified over-expression of mortalin in colorectal adenocarcinomas. Mortalin, also known as mitochondrial heat-shock protein 70 (mhsp 70), is involved in cell cycle regulation with important roles in cellular senescence and immortalization pathways. It is known to bind to and inactivate wild-type tumour suppressor protein p53 and influences the Ras-Raf-MAPK pathway. By immunostaining a colorectal cancer tissue microarray linked to a patient database, we further found that mortalin over-expression correlates with poor patient survival and, in multivariate analysis, is independent of standard prognostic variables (p = 0.04). Our findings demonstrate that mortalin over-expression may predict outcome in colorectal cancer and suggest that this protein is involved in colorectal neoplasia. Our experimental approach emphasises the analytical power of combining proteomics with tissue microarray analysis in the context of a well-defined tumour database. Copyright © 2004 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.