Tuberous sclerosis (TSC) is an autosomal dominant disorder characterized by hamartomas in one or more organs, including the brain, skin, heart and kidneys. Linkage studies have shown locus heterogeneity with one TSC gene mapped to chromosome 9q34 and a second to 16p13.3. The gene on 16p13.3, TSC2, has been cloned and shown to encode a 5.5 kb transcript that is widely expressed. To facilitate the search for mutations in the TSC2 gene product, tuberin, we have designed an RT-PCR-based assay system to scan the expressed coding region of the TSC2 gene in lymphoblasts. Using 34 overlapping PCR assays we performed single-strand conformation polymorphism analysis of DNA from 26 apparently sporadic TSC cases, two TSC families non-informative for linkage analysis and two confirmed chromosome 16-linked TSC families. Of the 60 chromosomes scanned, 14 showed abnormal SSCP mobility shifts. Using direct PCR sequencing we have identified five missense mutations, one 3 bp in-frame deletion and one 2 bp frameshift deletion, one nonsense mutation, one 29 bp tandem duplication and five silent nucleotide changes that are likely to be polymorphisms. There is no apparent clustering of mutations within TSC2. The diversity of mutation types argues that TSC2 may not act in a classic tumor suppressor fashion. In addition, we saw no specific correlation between the different mutations and clinical severity or expression. These data confirm that TSC2 is indeed the relevant gene, and that a substantial number of sporadic cases arise from mutations in the TSC2 gene.