Hepatitis delta virus (HDV) RNA detection was carried out, using a full-length HDV RNA probe, in serum of 43 patients with chronic HDV infection. Among them, 30 cases (70%) were HDV RNA-positive. With respect to other HDV markers, serum HDAg (detected by immunoblot) was found in 33 patients (77%) and IgM anti-HD in 29 (67%). A similar percentage of HDV RNA-positive patients with and without circulating hepatitis B virus (HBV) DNA (32.5 vs. 37%, respectively) was found. Antibodies against the human immunodeficiency virus (HIV) were detected in 15/43 subjects studied. The presence of HDV RNA was significantly higher (p < 0.05) in anti-HIV-seropositive cases (93%) than in the HIV-seronegative ones (57%). Moreover, simultaneous HDV and HBV replication was found more frequently (60 vs. 18%, p < 0.05) and at higher levels among the anti-HIV-positive patients than in the rest. In addition, in most of the anti-HIV-positive subjects, HDV RNA and HBV DNA were constantly positive during a whole year of follow-up.