Two-step monoclonal antibody tumor targeting using an avidin-biotin system has unique characteristics because of the high-affinity binding (1015 m-1) and the lower molecular weight ligands (avidin, streptavidin, or biotin) used as carriers of radioisotopes, toxins, or drugs. The distribution of radiolabeled streptavidin in a two-step targeting strategy was investigated in lung metastases of line 10 carcinoma in guinea plgs. The micro-distribution of administered D3 monoclonal antibody and 125I-labeled streptavidin in metastatic nodules was examined by immunohistochemistry and autoradiography, and the uptake was quantitated. With monoclonal antibody pretargeting, streptavidin was found mainly at the periphery of metastatic nodules 1.5 h after injection; it had penetrated deeper at 4 h and was approaching homogeneity in many of the tumor nodules at 24 h. These results indicate that streptavidin can penetrate into metastatic nodules more rapidly than can the antibody. The concentration of streptavidin in metastatic nodules 4 h after injection was 5.6 times higher for the pretargeted group than for the nonpretargeted group, and the pretargeting index was 4.7. Although the absolute uptake of streptavidin had decreased between 4 and 24 h, the metastasis:blood ratio had increased from 1.2 to 2.4. When compared with the animals injected with 125I-labeled D3 antibody alone, the pretargeted group achieved higher tumor:blood and tumor:lung ratios and a higher localization index at early times after injection of the radiolabeled species.