There is a lack of updated estimates of HIV-2 infection in Italy. Moreover, lack of standardized HIV-2 viral load (VL) and drug resistance tests challenges clinical practice. Among 2941 HIV-positive patients followed in our center (Brescia, Northern Italy), 220 (7.5%) were African at the beginning of the study period. We assessed a population of 151 HIV-Ab positive patients (141 of African origin), presenting for routine blood testing from January 2006 to May 2007. Those found infected with HIV-2 started an appropriate disease management with HIV-2 VL and genotypic drug resistance testing. Sixteen of 151 (10.6%) patients were positive for HIV-2. Of those 16 patients, 14 came from Africa. Among 7 experienced patients, 1 was responding to nelfinavir and 4 to lopinavir/ritonavir-containing regimens. Two patients were failing treatment: 1 patient was switched to a saquinavir/ritonavir-containing regimen and responded. The remaining patient switched to lamivudine + atazanavir + saquinavir + ritonavir did not respond, having had previous experience to multiple ineffective drugs, resulting in a very complex HIV-2 drug-resistance pattern. Accurate screening programs and integration of virological tools must be implemented urgently, given the high prevalence of HIV-2, particularly in immigrant patients.