Achaete-scute like (ASCL)2 is a basic helix-loop-helix transcription factor essential for the maintenance of proliferating trophoblasts during placental development. Using oligonucleotide microarrays we identified ascl2 as a gene significantly upregulated in colorectal adenocarcinomas (n=36 cancers, n=16 normals; 15-fold, P<0.0001). This finding was confirmed by quantitative reverse transcriptase (RT)-PCR on large intestinal cancers (n=29 cancers, n=16 normals; 10-fold, P<0.0001). In situ hybridization for ascl2 demonstrated expression at the base of small and large intestinal crypts (n=304), but in no other normal tissues excepting placenta. By in situ hybridization, 52-71% of colorectal adenomas (n=187), 50-73% of large (n=327) and 33-64% of small intestinal adenocarcinomas (n=124) were positive for ascl2 expression. Upregulation of murine ascl2 was also observed using oligonucleotide microarrays, quantitative RT-PCR and in situ hybridization on apcmin/+ and apc1638N/+ smad4-/+ tumours. Tumour cell lines stably transfected with LEF1(DN) or APC2, or transiently transfected with short-interfering RNA (siRNA) against beta-catenin showed a significant downregulation of ascl2. Colocalization of ascl2 with nuclear beta-catenin was observed in 73 small intestinal adenocarcinomas (P=0.0008) and apcmin/+ tumours. Preliminary in vitro data suggest ascl2 may promote progression through the G2/M cell cycle checkpoint. In summary, ascl2 is a putative regulator of proliferation that is overexpressed in intestinal neoplasia.