To describe the dynamic of HIV-1 prevalence in Italian childbearing women and to estimate the future incidence of pediatric AIDS due to vertical transmission, dried-blood specimens collected from a consecutive sample of newborns in all Italian regions were examined for the presence of anti-HIV-1 antibodies (HIV-Ab) after the routine neonatal screening program was completed. Of 555,722 blood samples collected and examined for HIV-Ab between 1990 and 1993, 550 (0.099%) were positive. Nationwide, the HIV seroprevalence decreased between 1990 and 1992 (0.124% in 1990, 0.100% in 1991, 0.085% in 1992), and increased, as compared with that in the previous year, in 1993 (0.096%). In an univariate analysis, HIV seroprevalence was positively was positively associated with being born in regions having higher AIDS cumulative incidence and in metropolitan areas, but negatively associated with year of delivery. In a multiple logistic regression analysis, only the AIDS cumulative incidence level of the delivery area and being born in a metropolitan area remained independently associated with HIV seroprevalence. Our results show significant geographical variation in the spread of HIV infection among childbearing women in Italy and provide useful indications to target prevention and care strategies for HIV-infected women and their children and to estimate the potential impact of implementing programs aimed at preventing vertical transmission of HIV infection.