Since the introduction in the mid-1980s, HIV testing has gradually improved both in terms of sensitivity and specificity. The so-called fourth generation of tests, combined HIV antigen/antibody assays, has now been introduced. This study compares three automated combined assays with older third-generation antibody assays in large-scale screening. Serum samples from routine screening of blood and plasma donors and clinical samples were investigated for specificity evaluation. Three fourth-generation combination assays from one manufacturer were compared with three older third-generation antibody assays from the same manufacturer. More than 40 000 samples per assay were included. For sensitivity, selected panels of confirmed HIV-1- and HIV-2-positive samples as well as seroconversion samples (HIV-1) from commercial panels and also from patients who appeared during the evaluation were used. The specificities of the fourth-generation tests were 99.91% (AxSYM), 99.95% (ARCHITECT) and 99.97% (PRISM) after repeated testing. Some specificity variation between reagent batches was observed. All HIV-1-positive samples were reactive by the three fourth-generation systems. HIV-1 seroconversion samples and panels were reactive earlier than by antibody-only tests. As for HIV-2 samples, AxSYM failed to detect one (n = 40), whereas PRISM and ARCHITECT detected all (n = 16 for PRISM and n = 52 for ARCHITECT). The new HIV antigen/antibody combination assay systems were found to have high sensitivity and specificity. The instruments provided a rational and easy way of testing at large scale.