The costimulatory CD40-CD40L pathway plays a critical role in the generation and maintenance of adaptive immune responses. Genetic interference of CD40-CD40L interactions strongly influences the onset and course in many autoimmune disease models including experimental autoimmune encephalomyelitis. We analysed the association of a single nucleotide polymorphism of the CD40 gene (C/T(-1)) in 287 patients with multiple sclerosis (MS) and 184 matched controls. No significant differences were found in the frequency of the C/T(-1) polymorphism between the patients with MS and the controls (53% vs 49%) or among different MS subtypes. Cell surface expression of CD40 did not differ within the different genotypes, but carriers of the T allele showed a trend for a lower stimulatory index compared with individuals with the CC genotype. Although these subtle differences indicate functional consequences in the immune stimulatory capabilities related to the CD40 C/T(-1) polymorphism, our population-based study found no association with disease susceptibility or disease course in MS.