The functional inactivation of the tumor susceptibility gene tsg101 in mouse NIH3T3 cells leads to cell transformation and the formation of metastatic tumors in nude mice. We cloned, mapped and sequenced the mouse tsg101 gene and further identified a processed pseudogene that is 98% identical to the tsg101 cDNA. Based on Northern blot analysis, tsg101 is expressed ubiquitously in mouse tissues. A comparison of the coding region of the mouse tsg101 gene with the human TSG101 cDNA revealed that both the mouse and human gene encode ten additional highly conserved amino acids at the N-terminus. Based on the mouse tsg101 genomic structure, we predicted four additional introns within the human TSG101 gene. Their location was confirmed using PCR and sequencing analysis. The presence of these so far unidentified introns now explains published data on aberrantly spliced mRNA products that were frequently observed in primary breast tumors. We show that a majority of shorter TSG101 transcripts are not the result of aberrant splicing events, but represent a fraction of true alternative splice variants. Finally, we examined tsg101 expression patterns during different stages of mammary gland development and in different transgenic mouse models for breast tumorigenesis.