After our initial reports 3-5 supporting a locus for tuberous sclerosis (TSC) on 9q, linkage analysis was undertaken in eight large multigeneration TSC families using nine polymorphic markers. Six of the markers were from the distal long arm of chromosome 9 and three from the long arm of chromosome 11. The data as a whole supported a TSC locus on distal 9q, the peak lod score on multipoint analysis being 3.77 6 cM proximal to the Abelson oncogene locus (ABL). However, analysis of 2-point lod scores using the HOMOG programs revealed significant evidence for genetic heterogeneity (p = 0.01), tight linkage to ABL being highly unlikely in one family. After exclusion of the unlinked family multipoint, analysis gave a peak lod score of 6.1 in the vicinity of ABL. The family unlinked to ABL showed no recombinants with two chromosome 11 probes, but it was too small to provide significant evidence for linkage. Genetic heterogeneity in TSC as demonstrated by this study will complicate efforts to clone the genes by reverse genetics and will severely hamper the use of linked probes for carrier detection and prenatal diagnosis.