A mutation leading to tuberous sclerosis was linked to the ABO blood group locus (9q34) on the long arm of chromosome 9. In an effort to confirm this assignment, nine multigenerational families with tuberous sclerosis, comprising 126 sampled individuals, were assessed for linkage of the ABO locus to tuberous sclerosis. Two-point linkage analysis and multilocus linkage analysis were used to evaluate linkage between tuberous sclerosis and the markers ABO, MCT136, and AblK2. Linkage of ABO to tuberous sclerosis was excluded for a distance of 20 centimorgans (cM) encompassing the region of the ABO locus. There was no evidence for genetic heterogeneity within this data set. Using 23 polymorphic markers, exclusion mapping demonstrated only a 1% probability that the tuberous sclerosis locus was on the distal short arm of chromosome 9 and provided no evidence in support of a tuberous sclerosis locus on the remainder of chromosome 9 including the area of the ABO locus.