The authors tested, by molecular hybridization, for hepatitis B virus DNA in serum specimens of 182 asymptomatic hepatitis B surface antigen (HBsAg) Greek carriers who were heterosexual partners of patients with acute hepatitis B (group A: 96 cases) or healthy subjects who were susceptible to hepatitis B (group B: 86 cases). The mean age (34.1 ± 10.4 vs. 33.9 ± 8.4 years) and the mean duration of sexual contact (6.9 ± 8.9 vs. 7.2 ± 6.3 years) were similar in the two groups of carriers. Hepatitis B virus DNA was detected significantly more frequently in group A than in group B (59.4% vs. 11.6%, p < 0.001). In particular, in group A, hepatitis B virus DNA was detected in 96.9% of hepatitis B e antigen (HBeAg)-positive and 41% of antibody to HBeAg (anti-HBe)-positive carriers. In contrast, in group B, hepatitis B virus DNA was identified in only 10.8% of anti-HBe-positive carriers (p < 0.001). These differences were especially significant in the young and middle-aged carriers (16–49 years old) and during the first four years of sexual contact. These data suggest that 1) there is a positive correlation between the presence of hepatitis B virus DNA in serum and the epidemiologic evidence of sexual transmission of hepatitis B virus, 2) hepatitis B virus DNA is a better indicator of infectivity than HBeAg/anti-HBe, and 3) the detection of hepatitis B virus DNA in serum probably identified carriers with high infectivity and potentially higher risk of transmitting hepatitis B virus to their sexual partners.