We studied annual human immunodeficiency virus (HIV) seroprevalence and incidence in a large number of intravenous drug users attending drug treatment centers in three Italian urban areas. We also evaluated risk factors for HIV seropositivity and for HIV seroconversion. The results showed that HIV prevalence and incidence are declining. HIV prevalence declined dramatically in study participants that were <25 years old. Prevalent HIV cases were associated with older age and longer duration of intravenous drug use; however, short duration of drug use increased the risk of seroconversion. The findings of our study suggest that comparing cross-sectional and longitudinal data contributes to a better understanding of the dynamics of the HIV epidemic among intravenous drug users.