The aims of this study were to observe trends in testing for HIV between 2004 and 2007 in one London, UK, hospital and to observe the seroprevalence of HIV within subgroups. Tests were grouped according to source and reason for testing. A total of 58,720 tests were considered (HIV-1 seroprevalence 0.9%). The majority (75.4%) of tests were performed as part of routine and opt-out protocols including antenatal and genitourinary (GU) screening. Among people specifically seeking HIV testing, the HIV seroprevalence was 3.5%. Medical specialties performed fewer tests but a high HIV seroprevalence was observed, including infectious disease (seroprevalence 4.4%) and other medical specialties (seroprevalence 3.4%). A small number of specialties performed few HIV tests. HIV testing was cost-effective in virtually all settings. This study suggests that more HIV tests could be performed, for example, in acute medicine, and training might increase the number of tests offered in some settings. The most effective way of increasing testing appears to be opt-out testing.