Tuberous sclerosis 2 (Tsc2) gene is the target of a germline insertion in the Eker rat model of inherited cancer susceptibility. This tumor suppressor gene, when mutated, gives rise to a spectrum of epithelial and nonepithelial neoplasms in the rat, as well as multisystem involvement of hamartomas in the human. In this study, we characterized the rat Tsc2 cDNA and found that it is highly homologous with the human gene, including a conserved rap1GAP catalytic domain. Sequence analysis of independent rat clones from a kidney cDNA library revealed distinct but related variants of the Tsc2 transcripts stemming from alternative splicing involving two noncontiguous exons within the translated region. The first of these, located at amino acids 947 to 990, gives rise to isoforms with or without the 129-bp exon. There exists another variant related to the use of a "cryptic" splice acceptor site in the downstream exon that results in an in-frame 3-bp deletion. A separate 69-bp exon encoding a novel serine-rich amino acid sequence (1272 to 1295) was also alternatively spliced. Together, we have found a minimum of four and potentially eight Tsc2 isoforms that are differentially expressed in a tissue-specific manner. These splice variants are highly conserved in the human gene, suggesting a possible functional role of these Tsc2 isoforms in various cell regulatory and developmental processes.