To facilitate detection of SV40 surface-associated tumor antigen (T-ag), conditions were established to surface label T-ag on intact cells by lactoperoxidase-catalyzed radioiodination (125I/LPO). SDS-PAGE analysis of anti-T immunoprecipitates of SV40-transformed and -infected cells labelled with 125I/LPO revealed the presence of iodinated T-ag. Several types of control experiments were employed to guarantee the surface specificity of the 125I/LPO labelling technique. When SV40-transformed mouse cells were surface labelled with lactoperoxidase and glucose oxidase immobilized on insoluble beads, a preparation less readily internalized than soluble enzymes, T-ag was iodinated. Selective immunoprecipitation of surface antigens demonstrated that lactoperoxidase did not iodinate internally localized T-ag. A reconstruction experiment in which an extract of SV40-infected cells was added to uninfected cells prior to surface labelling suggested that T-ag released from lysed cells did not adhere significantly to monolayer surfaces and become iodinated. Finally, systematic omission of reactants from the iodination reaction revealed that exogenous addition of lactoperoxidase and H2O2 was necessary to generate an iodinated T-ag, indicating that endogenous host cell reactants do not contribute significantly to the iodination of T-ag. 125I-labelled T-ag was detectable on the surface of SV40 tsA-infected cells at the nonpermissive temperature 24 h post infection, indicating that the tsA lesion does not prevent the interaction of T-ag with the cell surface. When 125I/LPO-labelled transformed or infected cells were chased for 2.5 h after labelling, iodinated T-ag was no longer associated with the cell monolayer but was immunoprecipitable from culture supernatants. Cultures from which labelled T-ag had been shed could then be relabelled with 125I/LPO and surface-associated T-ag was again detectable. These data suggest that surface-associated T-ag is continuously shed from the cell surface and is rapidly replaced in the membrane by intracellular T-ag.