We developed a transfection-neutralization assay for human immunodeficiency virus type 1 (HIV-1) infectious molecular clones. In this assay CD4 negative adherent cells, transfected in microtiter plates with fixed amounts of proviral DNA of molecular HIV-1 clones, are cocultivated with CD4 positive T cell lines or primary peripheral blood mononuclear cells (PBMC) in the presence of anti-HIV-1 sera or monoclonal antibodies (MAbs). Results obtained with this technique were reproducible and compared favorably with a conventional cell-free infection inhibition assay. The transfection-neutralization assay obviates the need for virus stock preparation and, therefore, is particularly suitable for the evaluation of HIV-1 clones with slow replication kinetics and of recombinant chimeric HIV-1 clones inclined to undergo additional mutations during stock preparation. The potential value of this assay for the analysis of the specificity of neutralizing sera and MAbs was demonstrated in experiments with V3 chimeric molecular clones.