We report the genomic cloning and sequence analysis of a novel and (so far) smallest coding exon of the human adenomatous polyposis coli (APC) gene. This additional exon of 54 nucleotides in length, designated APC exon 10A, is located 1.6 kb downstream from exon 10. It resides on a 0.5-kb genomic EcoRI restriction fragment. APC exon 10A is alternatively spliced and inserted in-frame into mature transcripts; it gives an APC protein with an additional 18 amino acids. This highly conserved exon is expressed in a tissue-independent fashion. APC exon 10A flanking sequences are presented so that this novel exon can be included in future mutation screening procedures.