PURPOSE: Surgical resection of pulmonary metastases is now a clinically accepted cancer therapy but its success depends on the accurate localization and removal of all tumor foci. To enhance the detection of pulmonary metastases during surgery, we developed an i.v. administered optical probe that uses a monoclonal antibody, Herceptin (trastuzumab), conjugated to a fluorophore, rhodamine green (RhodG), to specifically detect human epidermal growth factor receptor type 2 (HER2/neu)-expressing pulmonary lesions in an animal model of lung metastases. EXPERIMENTAL DESIGN: Pulmonary metastases were induced by i.v. injection of gene-transfected murine embryonic fibroblasts (3T3) cells in a murine model to produce a mixed population of HER2+ and HER2- tumors. To image these tumors, an anti-HER2 (Herceptin) or a control (HUT) complementarity-determining region-grafted antibody was conjugated to RhodG and injected i.v. into mice. Spectral fluorescence imaging was done after thoracotomy and images were correlated with gross and microscopic pathology to assess sensitivity and specificity. RESULTS: HER2+ tumors injected with Herceptin-RhodG were more fluorescent than either HER2- tumors or HER2+ tumors injected with HUT-RhodG at all time points. The maximal fluorescence signal in HER2+ tumors injected with Herceptin-RhodG was observed at 1 day postinjection. The tumors fluoresced primarily at the rim and not their center, reflecting the binding-site barrier that is commonly seen with high-affinity antibodies. CONCLUSION: A HER2-targeted optical imaging probe shows the ability to specifically enhance HER2+ pulmonary metastases but not HER2- pulmonary metastases. The high sensitivity and specificity of this probe is encouraging for the development of antigen-targeted optical probes to assist in the resection of pulmonary metastases.