The mammal Shc locus encodes three overlapping isoforms (46, 52, and 66 kDa) that differ in the length of their N-terminal regions. p46/p52Shc and p66Shc have been implicated, respectively, in the cytoplasmic propagation of growth and apoptogenic signals. Levels of p66Shc expression correlate with life span duration in mice. p46Shc and p52Shc are ubiquitously expressed, whereas p66Shc is expressed in a cell lineage-specific fashion. However, the mechanisms underlying the regulation of Shc protein expression are unknown. Here we report the identification of two alternative promoters, driving the transcription of two mRNAs coding for p46/p52Shc and p66Shc. We show that treatment with an inhibitor of histone deacetylases or with a demethylating agent results in induction of p66Shc expression in cells that normally do not express this isoform but leaves the levels of the two other isoforms unchanged. Moreover, analysis of the methylation pattern of the p66Shc promoter in a panel of primary and immortalized human cells showed inverse correlation between p66Shc expression and methylation density of its promoter. These results identify histone deacetylation and cytosine methylation as the mechanisms underlying p66Shc silencing in nonexpressing cells.