The high mortality and financial burden associated with prostate cancer can be partly attributed to a lack of sensitive screening methods for detection and staging of the disease. Guided by in silico docking studies using the crystal structure of PSMA, we designed and synthesized a series of PSMA-targeted (99m)Tc-chelate complexes for imaging PSMA-expressing human prostate cancer cells (LNCaP cell line). Of the six targeted radioimaging agents synthesized, three were found to bind LNCaP cells with low nanomolar affinity. Moreover, the same three PSMA-targeted imaging agents were shown to localize primarily to LNCaP tumor xenografts in nu/nu mice, with an average of 9.8 +/- 2.4% injected dose/g tissue accumulating in the tumor and only 0.11% injected dose/g tissue retained in the muscle at 4 h postinjection. Collectively, these high affinity, PSMA-specific radioimaging agents demonstrate significant potential for use in localizing prostate cancer masses, monitoring response to therapy, detecting prostate cancer recurrence following surgery, and selecting patients for subsequent PSMA-targeted chemotherapy.