A 65-year-old woman (C1I) and a 65-year-old man (C2I) contracted acute hepatitis C 40 or 42 years after marriage, respectively, in Japan. They had no discernible risk factors for acquiring hepatitis C virus (HCV) infection, except that they had monogamous sexual relationships with their spouses (C1S [66-year-old] with hepatocellular carcinoma and C2S [64-year-old] with liver cirrhosis, respectively) who were infected with HCV of the same genotype (1b) and had a high-titer HCV RNA in the serum (bDNA probe assay, 17 Meq/ml [C1S] and 15 Meq/ml [C2S]). The HCV isolates from Patients C1I and C1S and those from Patients C2I and C2S shared identity of 99.9% and 99.1%, respectively, in the 1,087-nucleotide (nt) sequence of the NS5B region, although these four isolates were only 91.7%-96.2% identical to the 94 reported genotype 1b isolates including those from Japanese patients. To confirm the high degree of genetic relatedness among ten HCV clones from each spouse within each pair of spouses, the E1 and E2 junctional region sequence (268 or 271 nt) including hypervariable region 1 (HVR-1) was analyzed. There was a close relationship between clones obtained from each spouse within each couple. Regarding the HVR-1 amino acid sequence, nine of the ten C1I clones were 100% identical with six of the ten C1S clones, and one each of the C2I and C2S clones differed by only one amino acid residue. This study indicates that two Japanese patients with acute hepatitis C had acquired HCV infection most probably by interspousal sexual transmission during a long-lasting marriage.