OBJECTIVE: To describe trends in HIV prevalence among injecting drug users in England and Wales between 1990 and 2003. METHODS: Analysis of surveillance data from voluntary unlinked anonymous cross-sectional surveys collecting oral fluid samples and behavioural information from injecting drug users recruited from both drug agency (n = 24,304) and community settings (n = 3628). RESULTS: HIV prevalence in England and Wales declined from 5.9% in 1990 to 0.6% in 1996 and then remained stable until 1999, after which it increased to 1.4% in 2003. Few HIV infections were detected among short-term injectors between 1994 and 1999, but in recent years prevalence among this group has increased. Other factors associated with higher odds of HIV infection were being recruited in London and from community settings, and ever having had a voluntary confidential HIV test. Incidence estimated through a force of infection model was 2.8% per annum among those injecting for less than a year in London between 1998 and 2003. CONCLUSIONS: These data suggest that incidence and prevalence of HIV may have increased, whereas other indicators suggest an increase in risk behaviour. It is critical that harm reduction measures are reinvigorated, and evolve in response to changes in drug use risk behaviours and policy.