Affinity iodination and cytotoxicity of hapten-substituted tumor cells were accomplished with an anti-hapten antibody conjugated to glucose oxidase (which generates H2O2) and lactoperoxidase (which iodinates cells in the presence of H2O2 and I-). Polynitrophenylated HeLa and Hep-2 cells, obtained by brief incubation with 2,4,6, trinitrophenyl (TNP)-sulfonic acid, were selectively iodinated and killed when treated briefly with an anti-TNP antibody-glucose oxidase conjugate (Ab-GO) and cultured in media containing lactoperoxidase (LP) and iodide. Cell killing was determined by three methods: 1) a microcytotoxicity assay, 2) the loss of cells prelabeled with 125IUdR and 3) the degree of incorporation of 125IUdR after treatment (postlabeling). Results with the three independent methods showed cytotoxicity, ranging from 19% to 98% in TNP-cells incubated with Ab-GO (0.6 to 57 µg/ml), LP (50 µg/ml), and iodide (20 µM). ε-DNP-lysine (100 µM) significantly reduced the amount of killing and minimal cytotoxicity (0 to 35%) was seen with unsubstituted cells or if Ab-GO, LP, or I- were omitted.