BACKGROUND: Improving blood safety without introducing nucleic acid testing in blood screening may be possible using antigen/antibody (Ag/Ab) combination assays, especially in resource-poor countries. STUDY DESIGN AND METHODS: To evaluate the potential reduction of human immunodeficiency virus (HIV)-transmitted infection using such an assay, a study was carried out aimed to compare the routine strategy for blood screening (S1), combining a rapid test (Determine HIV-1/-2, Inverness) and an enzyme immunoassay (human HIV-1/-2, Human Diagnostic) with an HIV Ag/Ab assay (S2, Genscreen ULTRA HIV Ag-Ab, Bio-Rad) in 2000 blood donations tested in Cameroon. Western blot and HIV RNA polymerase chain reaction were used to confirm the infection in reactive donors, and genotype was determined in HIV RNA-positive samples. RESULTS: Of the 2000 donors, 3.1% were positive with S1 and 4.3% with S2. Of the 97 samples positive with S1 and/or S2 tested for confirmation, 54.7% were positive, 38.1% indeterminate, and 7.2% negative. There were significant differences between S1 and S2 in terms of sensitivity (S1, 79.2%; S2, 100%), irrespective of the genotype, and in specificity (S1, 99.0; S2, 98.3%). The most frequent genotype (49%) was CRF02_AG. CONCLUSIONS: A testing strategy using Genscreen ULTRA HIV Ag/Ab could prevent 55 HIV transmissions per 10,000 donations. However, this would be at a cost of discarding 170 per 10,000 negative donations that would test false positive, showing that the implementation of new techniques in blood screening need an optimization before routine use. Using confirmatory assays, HIV Ab prevalence in blood donors in Cameroon was estimated at 2.65%.