tsg101 was identified as a tumor susceptibility gene by homozygous functional inactivation of allelic loci in mouse 3T3 fibroblasts. The human homologue was mapped at chromosome 11p15.1-2 and found to have intragenic deletion in 7 of 15 breast cancer specimens. To further confirm the relevance of defects in this gene to breast cancer, antibodies specific for the putative gene product were prepared and used to identify cellular TSG101 protein. The antibodies recognized a 46-kDa protein in human retinoblastoma WERI-27 cells labeled with [35S]methionine. This protein was not detected with preimmune sera. In cell fractionation studies, the 46-kDa protein cofractionating with glutathione S-transferase was found mainly in the cytoplasm. Similarly, when cells were immunostained with anti-TSG101 antibodies, fluorescence was localized in the cytoplasm of most of the cells. A full-size 46-kDa TSG101 protein was detected in a panel of 10 breast cancer cell lines and 2 normal breast epithelial cell lines with the same antibodies. Consistently, the full-length TSG101 mRNA was also detected in these breast cells using reverse transcription-PCR. These results indicate that homozygous intragenic deletion of TSG101 is rare in breast cancer cells.