To evaluate hepatitis B virus (HBV) risk group vaccination in Amsterdam, which started in 1998, we examined 342 reported acute HBV-cases and sequenced 85 DNA isolates. The reported number of cases declined from 214 in 1992-1997 to 128 in 1998-2003, due to a decline in injecting drug users (IDU) and their heterosexual partners. Phylogenetic analyses showed that after 1998, the IDU cluster nearly disappeared, probably due to a decline in injecting. Acute HBV remained stable among men having sex with men; given their increased sexual risk behavior, vaccination has probably prevented an increase in their acute infections. Currently, 48-72% of the people who should be included in the program are still susceptible to HBV.