We have studied 35 single nucleotide polymorphisms (SNPs) in the interferon (IFN) pathway to determine their contribution to multiple sclerosis (MS) and hepatitis C virus (HCV) infection. A total of 182 patients with MS, 103 patients with chronic hepatitis C, and 118 control subjects were enrolled in the study. Of the 35 SNPs studied, 3 were in IFN-alpha receptor (IFNAR-1), 10 in IFN-alpha/beta receptor (IFNAR-2), 9 in Stat1, 5 in Stat2, and 8 in IFN regulatory factor-1 (IRF-1). Compared to controls, Stat1 gene polymorphisms were significantly more frequent in MS patients (rs# 2066802 OR = 7.46, 95% CI = 2.22-25.10; rs# 1547550 OR = 1.69, 95% CI = 1.01-2.81) and in HCV patients (rs# 2066802 OR = 5.95, 95% CI = 1.55-22.81; rs# 1547550 OR = 2.30, 95% CI = 1.24-4.24). Also one IRF-1 gene SNP was associated with MS (rs# 2070721 OR = 2.05, 95% CI = 1.03-4.09), and four IRF-1 gene SNPs were associated with HCV infection (rs# 2070721 OR = 2.59, 95% CI = 1.23-5.43; rs# 2070723 OR = 4.8, 95% CI = 1.26-18.20; rs# 2070728 OR = 9.81, 95% CI = 1.21-79.4; rs# 2070729 OR = 3.6, 95% CI = 1.23-10.48; rs# 839 OR = 4.67, 95%CI = 1.29-16.87). Characteristic nucleotide combinations on single chromosomes (haplotype) generated block structures, including SNPs, that differed between patients and controls. Using a permutation test to detect differences in haplotype distribution between groups, the CCATTGA and the CCGAA haplotypes in the IRF-1 gene were more frequent in MS (p = 0.03) and in HCV patients (p = 0.001) than in controls. In conclusion, our data show that genetic variants in the IRF-1 and Stat1 genes of the IFN pathway are associated with MS and HCV infection.