One of the main genetic abnormalities associated with breast carcinogenesis is the loss of genetic material from chromosome arm 16q. Different groups have identified two regions (16q22.1 and 16q24-ter) that are frequently deleted in primary tumors, suggesting the presence of tumor suppressor genes in these regions. Little is known about the late stages of tumor progression in this respect, and we, therefore, analyzed biopsy specimens of breast cancer metastases for deletions in these critical regions of 16q. We examined fine needle cytopunctures from 24 metastases, each with lymphocyte DNA, for allelic imbalance on 16q by means of polymerase chain reaction (PCR) with 15 highly polymorphic markers. All the metastatic samples showed deletion of at least one informative locus on 16q. The loss of heterozygosity (LOH) pattern often indicated the loss of a complete long arm of chromosome 16 (13 cases); nevertheless, in the remaining 11 samples, partial LOH patterns were observed. A small region of overlap (SR02) in 16q22.1 was frequently involved, whereas another (SR01) in 16q24-ter was affected in only two cases. A third region of LOH in 16q22.2-q23.2 was found in 6/11 samples. These results suggest that at least three different regions are involved in allelic imbalance on chromosome arm 16q in breast cancer. Loss of material from the third region could be a major event in the genesis of metastases.