Background: Before implementing screening programmes for acute HIV infection in developing countries, key issues, including cost, feasibility and public health impact, must be determined. Objective: Fourth-generation enzyme immunoassay (EIA) was compared with HIV-1 RNA PCR for the detection of acute and early HIV infection in counselling and testing populations in Lima, Peru. Methods: Adults presenting for HIV testing at designated clinics in Lima-Callao, Peru were offered additional screening for acute HIV infection. All serum samples were tested with fourth-generation Ag/Ab EIA and confirmed by line immunoassay. Negative specimens were combined into 50-sample pools for HIV-1 RNA screening by PCR analysis in standard pooling algorithms. RNA-positive samples were retested with a third-generation EIA to evaluate the relative sensitivity of standard testing procedures. Results: Between 2007 and 2008 1191 participants were recruited. The prevalence of HIV infection was 3.2% (38/1191; 95% CI 2.2% to 4.2%) overall and 10.5% (25/237; 95% CI 6.6% to 14.5%) among men who reported sex with men (MSM). The prevalence of acute or recent HIV infection was 0.2% (95% CI 0% to 0.4%) overall and 0.8% (95% CI 0% to 2.0%) among MSM. Compared with third-generation EIA testing, both fourth-generation EIA and RNA PCR increased the rate of HIV case identification by 5.3% overall and by 8.0% within the subpopulation of MSM. Conclusions: Screening for acute HIV infection within Peru's resource-limited public health system was acceptable and detected a high prevalence of acute and recent HIV infection among MSM. Additional efforts are needed to screen for, and prevent, transmission of HIV among MSM in Peru during the acute seroconversion stage.