A new serological assay, the recombinant flow cytometric immunofluorescence assay (r-FIFA), was developed for the early detection of human immunodeficiency virus type 1 (HIV-1) antibodies by using recombinant insoluble forms of HIV-1 Gag-p45, Gag-gp41 chimeric protein, gp160, Po197 polyprotein as antigens and autologous carriers through flow cytometry. These recombinant proteins were expressed in insect cells by a baculovirus expression system. Eight anti-HIV-1 seroconversion panels, a low-titer anti-HIV-1 panel from Boston Biomedica Inc. (BBI), and three HIV-1 seroconversion specimens from the Provincial Health Laboratory of Ontario, Toronto, Ontario, Canada (PHL), were tested and analyzed by r-FIFA. In sensitivity comparisons between r-FIFA and tests licensed by the U.S. Food and Drug Administration, which were used to test all of the HIV-1 panels from BBI, detection of HIV-1 antibody by r-FIFA was on average greater than 20 days earlier than that by enzyme immunoassay. The sensitivity of r-FIFA has permitted the detection of HIV-1-specific immunoglobulin G (IgG), IgM, and IgA antibodies during seroconversion. A kinetic analysis of HIV-1 antibody production of r-FIFA has shown that either IgG or IgM, or both, can be detected, depending on the phase and type of the immune response in the HIV-1-infected individual. Both primary and secondary immune responses were observed during this period. The r-FIFA results suggest that implementation of r-FIFA may significantly reduce the "window" period from the time of infection to the time of seroconversion, with earlier detection of antibodies after initial infection. This would also make it possible for us to understand the immune response and the precise mechanisms of immunopathogenesis in the early period of HIV-1 infection.