To calculate the prevalence of hepatitis D virus (HDV) superinfection, antibody to HDV (anti-HD) was tested on admission in 696 hepatitis B virus (HBV) chronic carriers diagnosed between 1979 and 1992. Anti-HD was positive in 67 patients (9.6%), and it was more frequently detected in i.v. drug abusers (IVDA) (59/74, 79.7%) than in non-IVDA (8/622, 1.3%) (p = 0.0001). The prevalence of anti-HD was higher in HBV chronic carriers diagnosed between 1979 and 1985 (33/219, 15.1%) than in those diagnosed from 1986 to 1992 (34/477, 7.1%) (p < 0.001). However, these figures were not different when IVDA were analyzed alone; 29/34 (85%) and 30/40 (75%) (p = 0.4) IVDA diagnosed in both time frames were anti-HD-positive. Four hundred and thirty anti-HD-negative HBV carriers were prospectively followed, and serial determinations of anti-HD were made. Seroconversion to anti-HD was observed in only six patients (1.4%), and again the rate of seroconversion was higher in IVDA (5/10, 50%) than in non-IVDA (1/420, 0.2%) (p < 0.0001). These findings show that IVDA HBV chronic carriers are still an extremely high risk group for HDV superinfection in Spain and that this virus has little penetrance in other epidemiological categories of HBV carriers.