The ability of antibody induced by combination vaccination (human immunodeficiency virus type 1 (HIV-1LAV) gp160 live recombinant vaccinia virus priming followed by a booster injection with a baculovirus-expressed HIV-1LAV recombinant gp160 candidate vaccine) to bind to native and recombinant HIV-1 envelope glycoproteins was measured in 12 uninfected healthy adult volunteers. By using a flow cytometric indirect immunofluorescence assay (FIFA) to detect vaccine-induced antibody to native envelope glycoprotein expressed by target cells infected with HIV-1IIIB (infected-cell FIFA), sera from ten of 12 vaccinees before the rgp160 booster injection were positive, and all vaccinees were positive at higher levels after the rgp160 boost. Evidence for cross-reacting antibody to HIV-1MN, HIV-1RF and HIV-1CC expressed on infected cells was also present after the rgp160 booster injection. High titres of anti-rgp160 antibody were also measured in an enzyme-linked immunosorbent assay after the boost. None of the sera obtained immediately prior to the rgp160 booster injection, but all sera drawn after the boost, reacted with recombinant gp160 antigen bound to uninfected cell surfaces. The high anti-gp160 binding activity in these assays, the concomitant presence of positivity in infected-cell and rgp160-coated cell FIFA assays, and high anti-rgp160 antibody titre by ELISA in sera from recipients of this prime-boost vaccination regimen suggest that the live vector priming and rgp160 boosting strategy should be pursued in HIV-1 vaccine development.