We comprehensively screened CTLA4 for novel genetic variations in patients with MS. We studied genetic variations by association methods in a population-based sample of 122 sporadic patients with MS and 244 age-, gender- and ethnicity-matched controls, and by linkage and family-based association methods in 395 individuals from 59 American multiplex pedigrees with 141 affected individuals. Being homozygous for AT(8) (common) allele of the 3'(514) microsatellite (OR: 1.69; CI: 0.99-2.86) and for the common 5'(318)*C/E1(49)*A/3'(514*AT(8) haplotype (OR: 1.96; CI: 1.13-3.39) was associated with increased susceptibility to MS in Olmsted County. The genotype frequencies of other individual polymorphisms were not significantly different between cases and controls. A pooled analysis of association studies revealed an odds ratio of 1.28 (95% CI: 1.01-1.63; p=0.043) for 5'(-318)*C homozygotes and 1.28 (95% CI: 1.08-1.51; p=0.005) for the 3'(514)*AT(8) allele. We did not detect linkage with MS susceptibility in multiplex families. We did not find a strong association with age at onset, disease course or severity. CTLA-4 is associated with susceptibility to MS.