DNAs from 19 malignant human breast tumors and 2 benign fibroadenomas were analyzed for heterozygosity at 5 polymorphic loci on the short arm of chromosome 3. One homozygous deletion and one rearrangement were identified using probe D3S2 which maps to 3p14.3-3p21.1. This probe also detected novel hybridizing fragments of 2.0 kb and/or 3.4 kb in 6/18 (33%) of the malignant tumor samples that hybridized with the D3S2 probe following digestion with the 5'-methylcytosine-insensitive enzyme MspI. Comparisons of HpaII and MspI digestion showed that all but one of the tumor DNAs analyzed were hypermethylated. The two fibroadenoma DNAs were not as highly methylated and had hybridizing fragments of 3.4 kb after HpaII digestion. These malignant breast-tumor DNAs exhibit 3 mechanisms by which a tumor-suppressor gene hypothesized to reside at 3p14-3p21 could be inactivated: homozygous deletion, rearrangement and hypermethylation, and strongly implicate this 3p chromosome region in breast-tumor development.