Mutation of the APC tumor suppressor gene is one of the earliest events in the development of most colorectal tumors. The APC gene encodes multiple protein isoforms through a complicated pattern of expression and alternative splicing. The role that each isoform plays in cellular physiology is unknown, although the presence of some of these isoforms in postmitotic cells suggests a role in controlling cell growth or promoting differentiation. Three APC isoforms that differ in their amino-terminal domains were evaluated by gene transfer experiments using a colon cancer cell line that lacks functional APC. All three isoforms alter cellular morphology and affect cell growth by elongating the G1 phase of the cell cycle. The conventional APC and brain-specific APC isoforms suppress the tumorigenic phenotype of cultured cells, while the 0.3 APC isoform does not. In support of these experiments, BrdU incorporation as a marker for S-phase entry occurs at a higher level in transiently transfected cells with 0.3 APC when compared to cells transfected with the other isoforms. All three APC isoforms colocalize with microtubules and dramatically reduce beta-catenin activity to the same extent in transiently transfected cancer cells, suggesting that the different effects of each isoform on tumorigenesis may be nontranscriptional in origin.