Myf-3 is the human homologue of the murine Myo-D1 gene involved in muscle-cell differentiation. Using Southern blot analysis, we examined methylation of Myf-3 in histologically normal colonic mucosae, adenomas and carcinomas from a large series of patients with primary colorectal cancer. Hypermethylation of this gene in comparison with normal mucosa was observed in 88% of adenomas and in 99% of carcinomas. The pattern of Myf-3 methylation was similar in different areas of the same tumour, suggesting that methylation imbalances occur before the bulk of clonal-cell expansion. Significantly increased levels of Myf-3 methylation were observed in tumours which were more invasive, located in the proximal colon or from older patients. Patients whose tumours had extensive methylation showed a trend for shortened survival, though this was probably related to their being more invasive. Extensive methylation was significantly more frequent in tumours with microsatellite instability. Further work is required to determine whether the hypermethylation of Myf-3 observed in colorectal cancers is a specific alteration with functional significance or whether it reflects non-specific methylation imbalances occurring early during tumorigenesis.