Loss of heterozygosity within the short arm of chromosome 3 is a common molecular event in several types of solid tumors. In breast cancer, 3p loss of heterozygosity occurs in invasive tumor cells as well as in morphologically normal terminal ductal lobular units adjacent to carcinoma in some cases [G. Deng et al., Science (Wash. DC), 274: 2057-2059, 1996.]. The most frequent region of allelic loss at 3p24.3 in morphologically normal terminal ductal lobular units encompasses the thyroid hormone receptor beta1 (TRbeta1) gene. Here we have observed a variable degree of TRbeta1 promoter hypermethylation in all 11 cases of primary breast cancer examined. Moreover, hypermethylation occurred at the same CpG sites in nonmalignant tissue peripheral to carcinoma in 4 of 11 cases. The lack of TRbeta1 nuclear staining, a likely result of biallelic gene inactivation, was observed in 25% (22 of 85) of primary tumors. This is a first demonstration of promoter hypermethylation and a concurrent reduction of TRbeta1 transcripts in breast cancer cell lines, although specific CpG sites targeted for gene silencing remain to be determined. Gene expression was restored by treatment with 5-aza-deoxycytidine in such cases. The observation of early, frequent, and multiple mechanisms of TRbeta1 inactivation suggests a potential role for this gene in the suppression of breast tumorigenesis.