The DNA structure recognition protein MSH2 is an important protein in DNA mismatch repair due to its role in initiating the repair process. To examine the potential interactions between mismatch repair and base excision repair (BER) we have examined the effect of MSH2 knockdown on 6-thioguanine (6-TG), temozolomide (TMZ) and methylmethane sulphonate (MMS) induced toxicity in BER proficient and deficient cell lines. An shRNA expression vector containing Msh2 target sequences was designed and used to transfect mouse embryonic fibroblasts lacking either alkylpurine DNA N-glycosylase (Mpg) or endonuclease III homologue (Nth1). Significant knockdown of Msh2 gene expression was achieved with three different target sequences, with the highest level being shown by Msh2283. Clonal selection resulted in differing levels of knockdown in Mpg-/- cells: (69.0 ± 12.1% from 5 cell clones). Transfection of the Msh2283 sequence in Mpg+/+, Nth1+/+ and Nth1-/- cells resulted in average knockdowns of 45.1 ± 40.5% (3 clones), 58.0 ± 21.4% (5 clones) and 74.9 ± 14.8% (3 clones), respectively. Msh2 knockdown resulted in increased resistance to 6-TG in BER (MPG and NTH1) proficient and deficient cell lines with similar levels of knockdown (84 ± 4%) but increased resistance to TMZ only in Mpg+/+ and Nth1-/- cell lines and not Mpg-/- or Nth1+/+ cells as assessed by an MTT assay. Msh2 knockdown had no effect on sensitivity to MMS induced toxicity. In a clonogenic assay, Msh2 silenced Mpg+/+, Mpg-/-, Nth1+/+ and Nth1-/- cells were more resistant to TMZ. These results confirm previous studies showing that MSH2 is a key protein in influencing 6-TG and O6-methylguanine induced toxicity but also suggest that the effect of this protein depends upon the presence of other proteins in different DNA repair pathways. © 2009 Elsevier Ireland Ltd. All rights reserved.