Recently a new fourth generation microELISA for large scale blood screening has been described in which HIV p24 Ag detection was integrated in an anti-HIV-1/-2 and anti-HIV-1 group O assay based on a direct assay format: (Vironostika HIV Uni-Form II Ag/Ab (Van Binsbergen et al., (1998)). When compared to the third generation a-HIV assay (Vironostika HIV Uni-Form II plus O), the seroconversion window was narrowed with more than one week. A more precise window estimation based on seroconversion series with short sampling time intervals of up to 7 days, is described in parallel with that for the single HIV p24 Ag assay. It was found with 10 relevant seroconversion series that the HIV p24 Ag assay closes the seroconversion window with 6.2 days as compared to the 3rd generation a-HIV assay, while a window reduction of 4 days was found with Vironostika HIV Uni-Form II Ag/Ab. These seroconversion data show that with the new assay two-thirds of the current p24 Ag window is closed. There was no statistical difference in seroconversion sensitivity between the HIV p24 Ag and the new assay format. An extended evaluation of the new assay with subtype HIV-1 M and group O p24 Ag strains and with anti-HIV antibodies obtained from individuals infected with different HIV-1 subtypes showed that all subtypes of HIV-1 M and HIV-1 group O p24 antigen were detected as well as of HIV antibody.