The KLF6 gene encodes the Krüppel-like factor 6, a transcription factor that has been individualized as a tumor-suppressor gene involved in the regulation of cell proliferation and differentiation. Recently, high frequency (42%) of KLF6 mutations have been reported in colorectal cancers (CRC) as in prostate cancers, astrocytic gliomas and hepatocellular carcinomas. The aims of the study was to confirm the frequency of KLF6 mutations in a larger series of CRC than that previously published by using DNA extracted from frozen tissue samples, which have been proved to generate less mutational artefact than that extracted from formalin-fixed paraffin-embedded tissue samples, in order to compare KLF6 mutation frequency with that of other common genetic alterations and to determine genotype-phenotype correlations. Amplification and direct sequencing of KLF6 exon 2 of 76 CRC and matched normal frozen tissues was performed. Polymorphisms were observed in 14 cases, among which two (T35T and S116S) had not already been reported. No KLF6 somatic mutation was observed. Our data suggest a minor role of KLF6 mutation in colorectal carcinogenesis and underline the fact that the validity of sequence informations obtained from DNA extracted from formalin-fixed tissues may be limited.