PURPOSE: Diagnosis and resection of indeterminate pulmonary nodules (IPNs) is a growing challenge with increased utilization of chest computed tomography. Photoacoustic (PA) -guided surgical resection with local injection of indocyanine green (ICG) may have utility for IPNs that are suspicious for lung cancer. This preclinical study explores the potential of PA imaging (PAI) to detect ICG-labeled tumors. MATERIALS AND METHODS: ICG uptake by H460 lung cancer cells was evaluated in vitro. A phantom study was performed to analyze PA signal intensity according to ICG concentration and tissue thickness/depth using chicken breast. PA signals were measured up to 48 hours after injection of ICG (mixed with 5% agar) into healthy subcutaneous tissue, subcutaneous H460 tumors and right healthy lung in nude mice. RESULTS: Intracellular ICG fluorescence was detected in H460 cells co-incubated with ICG in vitro. The concentration dependence of the PA signal was logarithmic, and PA signal decline was exponential with increasing tissue depth. The PA signal of 2 mg/mL ICG was still detectable at a depth of 22 mm in chicken breast. The PA signal from ICG mixed with agar was detectable 48 hours post injection into subcutaneous tissue and subcutaneous H460 tumors in nude mice. Similar features of PA signals from ICG-agar in mice lung were obtained. CONCLUSION: The results from this preclinical study suggests that PAI of injected ICG-agar may be beneficial for identifying deeply located tumors. These features may be valuable for IPNs.