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Stored demographic data and HIV RT and protease sequences of 877 HIV patients attending for the first time the HIV/AIDS outpatient clinics of a reference Infectious Diseases centre in Northern Italy between 1999 and 2006 were stratified by 3-year spanning periods according to date of HIV infection. In the period 1980-1982, new infections were entirely caused by HIV-1 subtype B strains and were all diagnosed in injection drug users, 88.9% of whom were males. Injection drug users accounted for 12.8% of new infections in 2004-2006. The frequency of heterosexually-transmitted infections consistently increased until 2000 (from almost none to 51.5%) remaining stable afterwards. About half of heterosexual patients were females. HIV infections among homosexual men increased from 0% in 1980-1982 to 15-21% between 1998 and 2006. Overall, the frequency of non-B subtypes HIV strains increased from 0% in 1980-1982 to 20.3% in 2004-2006 with a greater impact in heterosexuals (from 0% in 1980-1982 to 30.5% in 2004-2006). In conclusion, a picture of the changing scenario of circulating HIV types and subtypes in a reference Infectious Diseases centre in Northern Italy over the past 26 years is provided. A progressive modification in risk factors for HIV infection and a significant increase in the frequency of non-B HIV strains were observed.