The product of the oncosuppressor adenomatous polyposis coli (APC) gene is involved in cell cycle arrest and apoptosis and its loss of function is associated with the development of colorectal carcinogenesis. Its transcriptional regulation seems rather complex and has not been completely elucidated up to now. In an attempt to identify the transcription start sites for the mouse Apc gene we have detected a novel transcript in mouse embryonic stem (ES) cells and colon tissue. This transcript contains an untranslated exon, whose flanking sequences exhibited strong promoter activity in transient transfection experiments. These results suggest that we have identified a novel promoter for the mouse Apc gene, localized about 40 kb upstream of the initiating methionine, which drives expression of the unique Apc transcript type detected in undifferentiated totipotent ES cells. Transcripts bearing the novel exon combined either with exon 1 or with exon 2 were detected in all mouse tissues tested.