Hereditary Non-Polyposis Colorectal Cancer (HNPCC) is associated with germline mutations in one of several MisMatch Repair (MMR) genes. An increasing proportion (20–25%) of the reported MSH2 variants consists of single amino-acid substitution with uncertain disease-causing significance. The present study was undertaken to functionally characterize 3 MSH2 nontruncating variants: p.Gly162Arg (c.484G>C), p.Asp167His (c.499G>C) and p.Arg359Ser (c.1077A>T). Missense alterations, were assessed in a human system for expression/stability and for the ability to heterodimerize with MSH6 and correctly localize into the nucleus. Functional assays results were correlated with clinical and genetic features indicative of HNPCC as MicroSatellite-Instability (MSI), abnormalities of MMR gene expression in tumour tissue (IHC) and familial history. p.Gly162Arg and p.Arg359Ser variants showed a clearly decreased expression level of the MutSá complex and were associated with an abnormal subcellular localization pattern, which can be suggestive of an incorrect MSH2/MSH6 heterodimerization. Functional analysis results were supported by MSI and IHC data and by familial cancer history. The subcellular localization assay, performed in a human expression system, classifies as pathogenetic two MSH2 nontruncating alterations providing a useful tool in genetic testing programs. © 2008 Wiley-Liss, Inc.